Method of transporting mesenchymal stem cells by means of a transporting solution and a method of administering stem cells to wounds

ABSTRACT

The present invention relates to a method of transporting a stem cell population, the method comprising transporting the stem cell population contacted with a liquid carrier. In addition, the present invention concerns a method of treating a subject having a disease, the method comprising topically administering a defined mesenchymal stem cell population to the subject, wherein the mesenchymal stem cell population is administered within about 96 hours from the time point the mesenchymal stem cell population has been harvested. Also concerned is a unit dosage comprising about 20 million cells, of about 15 million cells, of about 10 million cells, of about 5 million cells, of about 4 million cells, of about 3 million cells, of about 2 million cells, of about 1 million cells, of about 0.5 million cells, of about 0.25 million cells or of less than 0.25 million cells of a defined mesenchymal stem cell population.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 16/378,914 filed Apr. 9, 2019, now U.S. Pat. No.11,400,119, which claims the benefit of priority of U.S. ProvisionalApplication No. 62/655,198, filed Apr. 9, 2018, the content of which ishereby incorporated by reference it its entirety for all purposes.

A portion of the disclosure of this patent document contains materialwhich is subject to (copyright or mask work) protection. The (copyrightor mask work) owner has no objection to the facsimile reproduction byanyone of the patent document or the patent disclosure, as it appears inthe Patent and Trademark Office patent file or records, but otherwisereserves all (copyright or mask work) rights whatsoever.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ST26.XML format and is hereby incorporated by reference inits entirety. Said ST26.XML copy, created on Oct. 7, 2022, is namedSCH-4300-DV1_SeqListing and is 13 kilobytes in size.

FIELD OF THE INVENTION

The present invention relates to a method of transporting a stem cellpopulation, the method comprising transporting the stem cell populationcontacted with a liquid carrier. In addition, the present inventionconcerns a method of treating a subject having a disease, the methodcomprising topically administering a defined mesenchymal stem cellpopulation to the subject, wherein the mesenchymal stem cell populationis administered within about 96 hours from the time point themesenchymal stem cell population has been harvested. Also concerned is aunit dosage comprising about 20 million cells, of about 15 millioncells, of about 10 million cells, of about 5 million cells, of about 4million cells, of about 3 million cells, of about 2 million cells, ofabout 1 million cells, of about 0.5 million cells, of about 0.25 millioncells or of less than 0.25 million cells of a defined mesenchymal stemcell population.

BACKGROUND OF THE INVENTION

Mesenchymal stem cells isolated from the amniotic membrane of theumbilical cord and their wound healing properties have been firstreported in US patent application 2006/0078993 (leading to granted U.S.Pat. Nos. 9,085,755, 9,737,568 and 9,844,571) and the correspondingInternational patent application WO2006/019357. Since then, theumbilical cord tissue has gained attention as a source of multipotentcells; due to its widespread availability, the umbilical cord and inparticular stem cells isolated from the amniotic membrane of theumbilical cord (also referred to as “cord lining stem cells”) have beenconsidered as an excellent alternative source of cells for regenerativemedicine. See, Jeschke et al. Umbilical Cord Lining Membrane andWharton's Jelly-Derived Mesenchymal Stem Cells: the Similarities andDifferences; The Open Tissue Engineering and Regenerative MedicineJournal, 2011, 4, 21-27.

A subsequent study compared the phenotype, proliferation rate,migration, immunogenicity, and immunomodulatory capabilities of humanmesenchymal stem cells (MSCs) derived from the amniotic membrane of theumbilical cord (umbilical cord lining (CL-MSCs), umbilical cord blood(CB-MSCs), placenta (P-MSCs), and Wharton's jelly (WJ-MSCs) (Stubbendorfet al, Immunological Properties of Extraembryonic Human MesenchymalStromal Cells Derived from Gestational Tissue, STEM CELLS ANDDEVELOPMENT Volume 22, Number 19, 2013, 2619-2629. Stubbendorf et alconcluded that extraembryonic gestational tissue-derived MSC populationsshow a varied potential to evade immune responses as well as exertimmunomodulatory effects. The authors also found that CL-MSCs showed themost promising potential for a cell-based therapy, as the cells showedlow immunogenicity, but they also showed enhanced proliferative andmigratory potential so that future research should concentrate on thebest disease models in which CL-MSCs could be administered.

While mesenchymal stem cells of the amniotic membrane can easily beobtained using the protocol as described in US patent application2006/0078993 and International patent application WO2006/019357, itwould be of advantage for clinical trials with these cord lining MSC tohave at hand a method that allows to isolate a population of these cordlining MSC's that is highly homogenous and can thus be used for clinicaltrials.

Such a highly homogenous population of mesenchymal stem cells derivedfrom the amniotic membrane of the umbilical cord has been reported forthe first time in co-pending U.S. application Ser. No. 15/725,913, filed5 Oct. 2018 claiming priority to U.S. provisional application Ser. No.62/404,582 filed 5 Oct. 2017, the content of both of which isincorporated by reference herein in its entirety) and as well as inco-pending PCT application PCT/SG2017/050500 also filed 5 Oct. 2018claiming priority to U.S. provisional application No. 62/404,582 filed 5Oct. 2017 and meets the criteria for mesenchymal stem cells to be usedfor cellular therapy (also cf. the Experimental Section of U.S.application Ser. No. 15/725,913, Dominici et al, “Minimal criteria fordefining multipotent mesenchymal stromal cells. The InternationalSociety for Cellular Therapy position statement”, Cytotherapy (2006)Vol. 8, No. 4, 315-317, or Sensebe et al., “Production of mesenchymalstromal/stem cells according to good manufacturing practices: a,review”, Stem Cell Research & Therapy 2013, 4:66.

Stem cells such as the mesenchymal stem cells as described above arehowever typically not applied/administered to patients at the site wherethey are produced. Often a substantial amount of time passes in betweenthe harvesting of cells and their further utilization. There is thus aneed for the provision of certain carriers which keep cells viable andhealthy for a period of time typically used for transport or storage ofcells.

Accordingly, it is an object of the invention to provide a method oftransporting/storing stem cells, especially of a population ofmesenchymal stem cells from the amniotic membrane of umbilical cord thatmeets this need.

SUMMARY OF THE INVENTION

This object is accomplished by the methods and the unit dosage havingthe features of the independent claims.

In a first aspect, the invention provides a method of transporting astem cell population, the method comprising transporting said stem cellpopulation contacted with a liquid carrier, said liquid carriercomprising

i) Trolox;

ii) Na⁺;

iii) K⁺;

iv) Cl⁻;

v) H₂PO₄ ⁻;

vi) HEPES;

vii) Lactobionate;

viii) Sucrose;

ix) Mannitol;

x) Glucose;

xi) Dextran-40;

xii) Adenosine, and

xiii) Glutathione.

In a second aspect, the invention provides a method of treating asubject having a disease, the method comprising topically administeringa mesenchymal stem cell population as described herein to the subject,wherein the mesenchymal stem cell population is administered withinabout 96 hours from the time point the mesenchymal stem cell populationhas been harvested.

In a third aspect, the invention provides a unit dosage comprising about20 million cells, of about 15 million cells, of about 10 million cells,of about 5 million cells, of about 4 million cells, of about 3 millioncells, of about 2 million cells, of about 1 million cells, of about 0.5million cells, of about 0.25 million cells or of less than 0.25 millioncells of a mesenchymal stem cell population as described herein.

In a fourth aspect, the invention provides the use of a liquid carrierfor transporting a stem cell population, wherein the liquid carriercomprises

i) Trolox;

ii) Na+;

iii) K+;

iv) Cl−;

v) H2PO4−;

vi) HEPES;

vii) Lactobionate;

viii) Sucrose;

ix) Mannitol;

x) Glucose;

xi) Dextran-40;

xii) Adenosine, and

xiii) Glutathione.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the detaileddescription when considered in conjunction with the non-limitingexamples and the drawings, in which:

FIG. 1 shows the technical information sheet of Lonza for Dulbecco'smodified eagle medium, including the catalogue number of the DMEM usedfor the making of the illustrative example of a medium of the invention(PTT-6) in the Experimental Section;

FIG. 2 shows the technical information sheet of Lonza for Ham's F12medium;

FIG. 3 shows the technical information sheet of Lonza for DMEM:F12 (1:1)medium, including the catalogue number of the DMEM:F12 (1:1) medium usedfor the making of the illustrative example of a medium of the invention(PTT-6) in the Experimental Section;

FIG. 4 shows the technical information sheet of Life TechnologiesCorporation for M171 medium, including the catalogue number of the M171medium used for the making of the illustrative example of a medium ofthe invention (PTT-6) in the Experimental Section;

FIG. 5 shows the list of ingredients, including their commercialsupplier and the catalogue number that have been used in theExperimental Section for the making of the medium PTT-6.

FIGS. 6A-6C show the results of flow cytometry experiments in whichmesenchymal stem cells isolated from the umbilical cord have beenanalysed for the expression of the mesenchymal stem cell markers CD73,CD90 and CD105. For these experiments, mesenchymal stem cells wereisolated from umbilical cord tissue by cultivation of the umbilical cordtissue in three different cultivation media, followed by subculturing ofthe mesenchymal stem cells in the respective medium. The three followingculture media were used in these experiments: a) 90% (v/v/ DMEMsupplemented with 10% FBS (v/v), b) the culture medium PTT-4 describedin US patent application 2006/0078993 and the correspondingInternational patent application WO2006/019357 that consist of 90% (v/v)CMRL1066, and 10% (v/v) FBS (see paragraph [0183] of WO2006/019357 andc) the culture medium of the present invention PTT-6 the composition ofwhich is described herein. In this flow cytometry analysis, twodifferent samples of the cord lining mesenchymal stem cell (CLMC)population were analysed for each of the three used culture media. Theresults are shown in FIG. 6A to FIG. 6C.

FIG. 6A shows the percentage of isolated mesenchymal cord lining stemcells expressing stem cell markers CD73, CD90 and CD105 after isolationfrom umbilical cord tissue and cultivation in DMEM/10% FBS,

FIG. 6B shows the percentage of isolated mesenchymal cord lining stemcells expressing stem cell markers CD73, CD90 and CD105 after isolationfrom umbilical cord tissue and cultivation in PTT-4;

FIG. 6C shows the percentage of isolated mesenchymal cord lining stemcells expressing stem cell markers CD73, CD90 and CD105 after isolationfrom umbilical cord tissue and cultivation in PTT-6.

FIGS. 7A-7B show the results of flow cytometry experiments in whichmesenchymal stem cells isolated from the umbilical cord have beenanalysed for their expression of stem cells markers (CD73, CD90 andCD105, CD34, CD45 and HLA-DR (Human Leukocyte Antigen-antigen D Related)that are used for defining the suitability of multipotent humanmesenchymal stem cells for cellular therapy and compared to theexpression of these markers by bone marrow mesenchymal stem cells. Forthis experiment, the mesenchymal stem cells of the amniotic membrane ofthe umbilical cord were isolated from umbilical cord tissue bycultivation of the umbilical cord tissue in the culture medium of thepresent invention PTT-6 while the bone marrow mesenchymal stem cellswere isolated from human bone marrow using a standard protocol.

FIG. 7A shows the percentage of isolated mesenchymal cord lining stemcells that express the stem cell markers CD73, CD90 and CD105 and lackexpression of CD34, CD45 and HLA-DR after isolation from umbilical cordtissue and cultivation in PTT-6 medium;

FIG. 7B shows the percentage of isolated bone marrow mesenchymal stemcells that express CD73, CD90 and CD105 and lack expression of CD34,CD45 and HLA-DR.

FIG. 8 shows the experimental setup for comparison of differentcarriers. First mesenchymal stem cell population as described hereinwere outgrown in cell culture flasks. The amount of living mesenchymalstem cells was counted and then 2 million cells/vial were stored fordifferent periods of time in either PlasmaLyte-A or HypoThermosol™-FRS.After storage cells have been counted in sample of ≤50 μl daily for days1-5 (Total liquid withdrawal 250 μl) and checked for viability bystaining the cells with Trypan blue. Further, on days 1, 3 and 5 sample≤80 μl were taken and analyzed. In addition, the supernatant wasobtained and frozen. Then PDGF-AA, PDGF-BB, VEGF, IL-10, Ang-1, HGF andTGFβ1 were measured by FLEXMAP 3D system.

FIG. 9 summarizes viability data. As can be seen from the left-handgraph, 73% of the total number of cells (about 95%) when the storingstarted were still viable 7 days after storage in HypoThermosol™. On thecontrary after 7 days of storage in PlasmaLyte-A only 42% of the totalnumber of cells (about 94%) when the storage started were still viable.All counts were based on duplicate readings that are within 10% of oneanother (following SOP CR D2.600.1). During counting, cells stored inHypoThermosol™ were noticeably smaller with smooth and defined edges. Bycontrast, cells in Plasmalyte-A appeared in a range of sizes.HypoThermosol™ noticeably supports membrane integrity and presumablysurvival over a 6 day timespan. Similar results are also shown in thegraph of the right-hand side.

FIG. 10 shows the results obtained when measuring the cell diameter ofcells. The mesenchymal stem cell population as described herein whenkept in HypoThermosol™ are narrower in diameter range when compared tocells kept in PlasmaLyteA. Comparison took place after 3 days ofstorage.

FIG. 11 shows the TGFß1 concentration in supernatant from themesenchymal stem cell population as described herein stored inHypoThermosol™ or PlasmaLyte-A after 48 hrs of storage. As can be seenfrom the graph on the right-hand side, cells secrete about as much TGFß1when stored in HypoThermosol™ as when stored in PlasmaLyte-A. Ingeneral, over time, the amount of secreted TGFß1 decreased (graph on theright hand side).

FIG. 12 shows control experiments. Here, the PDGF-BB concentrations weremeasured in supernatant from mesenchymal stem cell populations asdescribed herein stored in HypoThermosol™ or PlasmaLyte-A for 48 hrs.Since PDGF-BB are not normally secreted by the mesenchymal stem cellpopulation as described herein, no PDGF-BB were detectable in anysample.

FIG. 13 shows control experiments. Here, the IL-10 concentrations weremeasured in supernatant from mesenchymal stem cell populations asdescribed herein stored in HypoThermosol™ or PlasmaLyte-A for 48 hrs.Since IL-10 are not normally secreted by the mesenchymal stem cellpopulation as described herein, no IL-10 were detectable in any sample.

FIG. 14 shows the VEGF concentration in supernatant from mesenchymalstem cell populations as described herein stored in HypoThermosol™ orPlasmaLyte-A for 48 hrs. As can be seen from the graph on the right-handside, cells secrete about as much VEGF when stored in HypoThermosol™ orPlasmaLyte-A on day 0. On day 1 and 5 cells secreted more VEGF whenstored in PlasmaLyte-A. Notably, when stored for 3 days cells secretedmore VEGF when stored in HypoThermosol™ than when stored inPlasmaLyte-A. Thus, HypoThermosol™ outperforms PlasmaLyte-A by day 3 ofstorage. The more VEGF detected, the healthier is the culture. Thus, bysecreting more VEGF after 3 days storage in HypoThermosol™ than whenstored in PlamsaLyte-A, cells were healthier in HypoThermosol™ than inPlamsaLyte-A. From 5 days, storage in PlasmaLyte seems to become morefavourable, because at the time point cells stored in PlasmaLyte-Asecreted more VEGF. In general, over time, the amount of secreted VEGFdecreased (graph on the right hand side).

FIG. 15 shows the PDGF-AA concentration in supernatant from mesenchymalstem cell population as described herein stored in HypoThermosol™ orPlasmaLyte-A for 48 hrs. As can be seen from the graph on the right-handside, cells secrete about as much PDGF-AA when stored in HypoThermosol™as when stored in PlasmaLyte-A on day 0. On day 1 and 5 cells secretedmore PDGF-AA when stored in PlasmaLyte-A. Notably, when stored for 3days, cells secreted more PDGF-AA when stored in HypoThermosol™ thanwhen stored in PlasmaLyte-A. Thus, cells stored in HypoThermosol™ arehealthier than cells stored in PlasmaLyte-A after 3 days of storage.From 5 days of storage onwards, PlasmaLyte seems to become a morefavourable carrier, because at the time point cells stored inPlasmaLyte-A secreted more PDGF-AA. In general, over time, the amount ofsecreted PDGF-AA decreased (graph on the right hand side).

FIG. 16 shows the Ang-1 concentration in supernatant from mesenchymalstem cell populations as described herein stored in HypoThermosol™ orPlasmaLyte-A for 48 hrs. As can be seen from the graph on the right-handside, cells secrete about as much Ang-1 when stored in HypoThermosol™ orPlasmaLyte-A on day 0 and 3. On day 5 cells secreted more Ang-1 whenstored in PlasmaLyte-A. Noticably, when stored for 1 day, cells secretedmuch more Ang-1 when stored in HypoThermosol™ than when stored inPlasmaLyte-A. Thus, cells stored in HypoThermosol™ seem to be healthierthan when stored in PlasmaLyte-A for at least 48 hrs until day 3 ofstorage. From day 5, PlasmaLyte seems to become a more favourablecarrier, because at this time point cells stored in PlasmaLyte-Asecreted more Ang-1. In general, over time, the amount of secreted Ang-1decreased (graph on the right hand side).

FIG. 17 shows the HGF concentration in supernatant from mesenchymal stemcell populations as described herein stored in HypoThermosol™ orPlasmaLyte-A after 48 hrs of storage. As can be seen from the graph onthe right-hand side, cells secrete about as much HGF when stored inHypoThermosol™ than when stored in PlasmaLyte-A on day 0. On day 3 and 5cells secreted more HGF when stored in PlasmaLyte-A. Notably when storedfor 1 day, cells secreted much more HGF when stored in HypoThermosol™than when stored in PlasmaLyte-A. Thus, cells stored in HypoThermosol™seem to be healthier than cells stored in PlasmaLyte-A between at least1 day (48 hrs) until 3 days of storage. From 3 days onwards PlasmaLyte-Aseems to become a more favourable carrier, because at the time points 3and 5 days, cells stored in PlasmaLyte-A secreted more HGF. In general,over time, the amount of secreted HGF decreased (graph on the right handside).

FIG. 18 are photographs obtained from a preclinical study with themesenchymal stem cell population of the present invention in pigs. Thepigs were rendered diabetic with 120 mg/kg streptozotocin and allowed torecover for 45 days prior to creating six 5 cm×5 cm full thicknesswounds on their backs. Pigs (n=2) were treated twice weekly with 10⁵human mesenchymal stem cell population as described herein per cm² for 4weeks. The two control pigs were treated with PBS. Wounds werephotographed on postoperative day 0 (PODay 0) and every seven days untilpostoperative Day 35. The wounds were analyzed for surface area size byImageJ. By Day 35, the addition of mesenchymal stem cell population asdescribed herein had resulted in closure of 10 of 12 diabetic wounds(83%), compared to only 3 of 12 (25%) of the PBS-treated control wounds.The rate of wound healing was 0.8 cm²/day with the mesenchymal stem cellpopulation as described herein compared to 0.6 cm²/day in the controlanimals, an improvement of 33%.

FIG. 19 datasheet of Trolox available from Tocris.

FIG. 20 shows the datasheet of NaCl available from Sigma Aldrich.

FIG. 21 shows the datasheet of KH₂PO₄ available from Sigma Aldrich.

FIG. 22 shows the datasheet for HEPES from Sigma Aldrich.

FIG. 23 shows the product sheet for sodium lactobionate fromCOMBI-BLOCKS.

FIG. 24 shows the product sheet for sucrose from Sigma Aldrich.

FIG. 25 shows the product sheet for mannitol from avantor.

FIG. 26 shows the product sheet for glucose from Sigma Aldrich.

FIG. 27 shows the product sheet for Dextran-40 from Sigma Aldrich.

FIG. 28 shows the product sheet for adenosine from Sigma Aldrich.

FIG. 29 shows the product sheet for glutathione from Sigma Aldrich.

FIG. 30 shows the product sheet for HypoThermosol™-FRS (HTS-FRS) fromSTEMCELL Technologies.

DETAILED DESCRIPTION OF THE INVENTION

As explained above, in a first aspect the invention is directed to amethod of transporting/storing a stem cell population, the methodcomprising transporting/storing said stem cell population contacted witha liquid carrier, said liquid carrier comprising

i) Trolox;

ii) Na⁺;

iii) K⁺;

iv) Cl⁻;

v) H₂PO₄ ⁻;

vi) HEPES;

vii) Lactobionate;

viii) Sucrose;

ix) Mannitol;

x) Glucose;

xi) Dextran-40;

xii) Adenosine, and

xiii) Glutathione.

It has been surprisingly found in the present application that using aliquid carrier as described herein and in particular a liquid carriersuch as HypoThermosol™ leads to a superior survival of stem cellscompared to other pharmaceutically approved carriers such as e.g.PlasmaLyte®. For example, after 7 days of storage of a mesenchymal stemcell population as described herein in HypoThermosol™ about 70% of thecells were still viable. On the contrary, after 7 days of storage inPlasmaLyte® only about 40% of the cells were still viable (see Examples,when measured with a hemocytometer). Thus, using a liquid carrier asdescribed herein allows the transport/storage of stem cells over aperiod of time without substantial loss of the viability of cells. Inparticular, storage in HypoThermosol™ for shorter time period of 3 daysor less seems to be especially beneficial, since the stem cells ingeneral secreted more factors than after storage in PlasmaLyte-A asdescribed in the Experimental Section in detail.

When used herein the term ‘transport’ or ‘transporting’ any transport ismeant. Such transport may be performed with any vehicle, such as car,train, and airplane or by a person carrying/transporting a containercomprising the stem cells contacted with the liquid carrier from oneplace to another place. In one embodiment, transporting is carried outfrom the place of production of the stem cell population of interest tothe place of stem cell administration (for example, the GMP facility inwhich a stem cell population of interest is produced to the site ofadministration of the stem cell population, for example, a clinic or adoctor's office). It is however also envisioned that the term‘transporting relates to a storage of cells at the same place for aperiod of time. For example, stem cells may be stored after harvestuntil their application to a subject at one place. The container inwhich the stem cells can be stored or transported can be any containersuitable for the method of the present invention.

The transporting/storing can be performed for any period of time. Forexample, the transporting/storing can be performed for about 7 days orless. It is also envisioned that the transporting/storing can beperformed for about 6, 5, 4, 3, 2, 1, day(s) or less. It can thus bethat the transporting/storing is performed for about 48 hours or about24 hours or less.

It is also contemplated that the transporting/storing is performed atany temperature suitable for the method of the present invention. Forexample, the transporting/storing can be performed at a temperature ofabout −5° C. to about 15° C. It is therefore also envisioned that thetransporting/storing can be performed at a temperature of about 2° C. toabout 8° C. The transporting can also be carried out at a temperature ofmore than about −5° C., more than about −10° C., more than about −15°C., or more than about −20° C. Further it is envisioned thattransporting/storing can be performed at a temperature of below 20° C.,below 18° C., below 15° C., below 12° C. or below 10° C.

The method of the present invention also envisions that the stem cellpopulation is stored or transported in any suitable concentration. Thestem cell population may, for example, be transported/stored in aconcentration of about 70 million cells per 1 ml carrier, of about 60million cells million cells per 1 ml carrier, of about 50 million cellsper 1 ml carrier, of about 40 million cells per 1 ml carrier, of about30 million cells per 1 ml carrier, of about 20 million cells per 1 mlcarrier, of about 10 million cells per 1 ml carrier, of about 5 millioncells per 1 ml carrier, of about 4 million cells per 1 ml carrier, ofabout 3 million cells per 1 ml carrier, of about 2 million cells per 1ml carrier, of about 1 million cells per 1 ml carrier, of about 0.5million cells per 1 ml carrier, of about 0.1 million cells per 1 mlcarrier or of less than 0.1 million cells per 1 ml carrier. Therefore,the stem cell population can be transported/stored in a concentration ofabout 10 million cells per ml carrier to about 1 million cells per 1 mlcarrier.

The method of the present invention concerns the transporting/storing ofstem cells. In principle, any stem cell can be used in the method of thepresent invention. One characterizing feature of stem cells is theirability to self-renew. ‘Self-renewal’ is the ability to go throughnumerous cell cycles of cell division while maintaining theundifferentiated state. Methods for testing if a cell has the capacityto self-renew are known to the skilled artisan. For example,self-renewal may be tested by passaging the cells over more than 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30 or more passages. Passaging includes splitting of the cells beforere-plating them as a single cell suspension. A further characteristic ofstem cells is their multipotency or pluripotency as will also bedescribed elsewhere herein. In principle, multipotency or pluripotencycan be tested by differentiating said stem cells into differentlineages.

In particular, the stem cell population used in the method of thepresent invention can be an embryonic stem cell population, an adultstem cell population, a mesenchymal stem cell population or an inducedpluripotent stem cell population.

As used herein an “embryonic stem cell population” is a “pluripotentstem cell population”. A pluripotent cell when referred to hereinrelates to a cell type having the capacity for self-renewal, and thepotential of differentiation into different cell types. Pluripotent stemcells can differentiate into nearly all cells, i.e. cells derived fromany of the three primary germ layers: ectoderm, endoderm, and mesoderm.The term pluripotent stem cell also encompasses stem cells derived fromthe inner cell mass of an early stage embryo known as a blastocyst.Notably, recent advances in embryonic stem cell research have led to thepossibility of creating new embryonic stem cell lines without destroyingembryos, for example by using a blastomere biopsy-based technique, whichdoes not interfere with the embryo's developmental potential(Klimanskaya (2006) “Embryonic stem cells from blastomeres maintainingembryo viability.” Semin Reprod Med. 2013 January; 31(1):49-55).Furthermore, a large number of established embryonic stem cell lines areavailable in the art. Thus, it is possible to work with embryonic stemcells without the necessity to destroy an embryo. The pluripotent stemcells can be embryonic stem cells, which have not been obtained via thedestruction of a human embryo. Thus, the pluripotent stem cells areembryonic stem cells obtained from an embryo, without the destruction ofthe embryo.

As used herein an “adult stem cell population” is a multipotent stemcell population. A multipotent stem cell population can give rise arestricted number of cell types, therefore they are somatic faterestricted. For example, a neural stem cell can give rise to bothneuronal and glial cells. Adult stem cells have the capability toself-renew and may be obtained from any suitable source. For example,adult stem cells may be obtained from bone marrow, peripheral blood,brain, spinal cord, dental pulp, blood vessels, skeletal muscle,epithelia of the skin and digestive system, cornea, retina, liver, orpancreas.

The stem cell population used in the method of the present invention mayalso be a mesenchymal stem cell population. In this context, it is notedthat the culture medium described herein (e.g. PTT-6) allows theisolation of a mesenchymal stem cell population (also referred herein as“mesenchymal stem cells”) from the amniotic membrane under conditionsthat allow cell proliferation of the mesenchymal stem/progenitor cellswithout differentiation of the mesenchymal stem/progenitor cells. Thus,after isolation of the mesenchymal stem cells from the amniotic membraneas described herein the isolated mesenchymal stem/progenitor cellpopulation has the capacity to differentiate into multiple cell types asdescribed in US patent application 2006/0078993, U.S. Pat. No.9,085,755, International patent application WO2006/019357, U.S. Pat. No.8,287,854 or WO2007/046775, for instance. As described in US patentapplication 2006/0078993, for example, the mesenchymal stem cells of theamniotic membrane of the umbilical cord have a spindle shape, expressthe following genes: POU5f1, Bmi-1, leukemia inhibitory factor (LIF),and secrete Activin A and Follistatin. The mesenchymal stem cellsisolated in the present invention can, for example, be differentiatedinto any type of mesenchymal cell such as, but not limited to,adipocytes, skin fibroblasts, chondrocytes, osteoblasts, tenocytes,ligament fibroblasts, cardiomyocytes, smooth muscle cells, skeletalmuscle cells, mucin producing cells, cells derived from endocrine glandssuch as insulin producing cells (for example, β-islet cells) orneurectodermal cells. The stem cells isolated in accordance with themethod described herein can be differentiated in vitro in order tosubsequently use the differentiated cell for medical purposes. Anillustrative example of such an approach is the differentiation of themesenchymal stem cells into insulin producing β-islet cells which canthen be administered, for example by implantation, to a patient thatsuffers from an insulin deficiency such as diabetes mellitus (cf. alsoWO2007/046775 in this respect). Alternatively, the mesenchymal stemcells described herein can be used in their undifferentiated state forcell-based therapy, for example, for wound healing purposes such astreatment of burns or chronic diabetic wounds. In these therapeuticapplications the mesenchymal stem cells of the invention can eitherserve to promote wound healing by interacting with the surroundingdiseased tissue or can also differentiate into a respective skin cell(cf., again WO2007/046775, for example).

In this context, it is noted that the mesenchymal stem cell populationdescribed herein can be isolated and cultivated (i.e. are derived) fromany umbilical cord tissue as long as the umbilical cord tissue containsthe amniotic membrane (which is also referred to as “cord lining”).Accordingly, the mesenchymal stem cell population can be isolated from(pieces of) the entire umbilical cord as described in the Experimentalsection of the present application. This umbilical cord tissue may thuscontain, in addition to the amniotic membrane, any othertissue/component of the umbilical cord. As shown, for example, in FIG.16 of US patent application 2006/0078993 or International patentapplication WO2006/019357, the amniotic membrane of the umbilical cordis the outermost part of the umbilical cord, covering the cord. Inaddition, the umbilical cord contains one vein (which carriesoxygenated, nutrient-rich blood to the fetus) and two arteries (whichcarry deoxygenated, nutrient-depleted blood away from the fetus). Forprotection and mechanical support these three blood vessels are embeddedin Wharton's jelly, a gelatinous substance largely ofmucopolysaccharides. Accordingly, the umbilical cord tissue used hereincan also comprise this one vein, the two arteries and the Wharton'sjelly. The use of such an entire (intact) section of the umbilical cordhas the advantage that the amniotic membrane does not need to beseparated from the other components of the umbilical cord. This reducesthe isolation steps and thus makes the method described herein, simpler,faster, less error prone and more economical—which are all importantaspects for the GMP production that is necessary for therapeuticapplication of the mesenchymal stem cells. The isolation of themesenchymal stem cells can thus start by tissue explant, which may befollowed by subsequent subculturing (cultivation) of the isolatedmesenchymal stem cells if greater amounts of the mesenchymal stem cellsare desired, for example, for use in clinical trials. Alternatively, itis also possible to first separate the amniotic membrane from the othercomponents of the umbilical cord and isolate the mesenchymal cord liningstem cells from the amniotic membrane by cultivation of the amnioticmembrane in a culture medium e.g. PTT-6. This cultivation can also becarried out by tissue explant, optionally followed by subculturing ofthe isolated mesenchymal stem cells. In this context, the term “tissueexplant” or “tissue explant method” is used in its regular meaning inthe art to refer a method in which a tissue, once being harvested, or apiece of the tissue is being placed in a cell culture dish containingculture (growth) medium and by which over time, the stem cells migrateout of the tissue onto the surface of the dish. These primary stem cellscan then be further expanded and transferred into fresh dishes throughmicropropagation (subculturing) as also described here. In this context,it is noted that in terms of production of the cells for therapeuticpurposes, in the first step of isolating the amniotic membranemesenchymal stem cells from the umbilical cord, a master cell bank ofthe isolated mesenchymal stem cells is obtained, while with thesubsequent subculturing, a working cell bank can be obtained. Inparticular embodiments, the stem cell population thus is a mesenchymalstem cell population.

The mesenchymal stem cell population may be isolated from the amnioticmembrane of the umbilical cord by a method comprising cultivatingumbilical cord tissue in a culture medium comprising DMEM (Dulbecco'smodified eagle medium), F12 (Ham's F12 Medium), M171 (Medium 171) andFBS (Fetal Bovine Serum). Using such a medium provides for the isolationof a mesenchymal stem cell population from the amniotic membrane of theumbilical cord of which more than 90%, or even 99% or more of the cellsare positive for the three mesenchymal stem cell markers CD73, CD90 andCD105 while at the same these stem cells lack expression of CD34, CD45and HLA-DR (see the Experimental Section), meaning 99% or even morecells of this population express the stem cell markers CD73, CD90 andCD105 while not expressing the markers CD34, CD45 and HLA-DR. Such anextremely homogenous and well defined cell population has been reportedfor the first time in co-pending U.S. application Ser. No. 15/725,913,filed 5 Oct. 2018 claiming priority to U.S. provisional application Ser.No. 62/404,582 filed 5 Oct. 2017, the content of both of which isincorporated by reference herein in its entirety) and as well as inco-pending PCT application PCT/SG2017/050500 also filed 5 Oct. 2018claiming priority to U.S. provisional application No. 62/404,582 filed 5Oct. 2017 and is the ideal candidate for clinical trials and cell basedtherapies since, this stem cell population for example, fully meets thecriteria generally accepted for human mesenchymal stem cells to be usedfor cellular therapy as defined, for example, by Dominici et al,“Minimal criteria for defining multipotent mesenchymal stromal cells.The International Society for Cellular Therapy position statement”,Cytotherapy (2006) Vol. 8, No. 4, 315-317, Sensebe et al., “Productionof mesenchymal stromal/stem cells according to good manufacturingpractices: a, review”, Stem Cell Research & Therapy 2013, 4:66), Vonk etal., Stem Cell Research & Therapy (2015) 6:94, or Kundrotas Acta MedicaLituanica. 2012. Vol. 19. No. 2. P. 75-79. Also, using a bioreactor suchas a Quantum Cell Expansion System, it is possible to obtain highnumbers of mesenchymal stem cells such as 300 to 700 million mesenchymalstem cells per run (see also the Experimental Section). Thus, thepresent invention allows transporting/storing amounts of stem cells thatare needed for therapeutic applications, such as their use in woundhealing, in a cost efficient manner. In addition, all components usedfor making the culture medium of the present invention are commerciallyavailable in GMP quality. Accordingly, the present invention opens theroute to transport/store a GMP produced and highly homogenousmesenchymal stem cell population from the amniotic membrane of theumbilical cord.

Thus, in some embodiments the mesenchymal stem cell population is anisolated mesenchymal stem population of the amniotic membrane of theumbilical cord. It is further envisioned that at least about 90% or morecells of the isolated mesenchymal stem cell population express each ofthe following markers: CD73, CD90 and CD105. For example, at least about91% or more, about 92% or more, about 93% or more, about 94% or more,about 95% or more, about 96% or more, about 97% or more, about 98% ormore about 99% or more cells of the isolated mesenchymal stem cellpopulation express each of CD73, CD90 and CD105. Additionally oralternatively, at least about 90% or more, about 91% or more, about 92%or more, about 93% or more, about 94% or more, about 95% or more, about96% or more, about 97% or more, about 98% or more about 99% or more ofthe isolated mesenchymal stem cells lack expression of the followingmarkers: CD34, CD45 and HLA-DR (Human Leukocyte Antigen-antigen DRelated).

The marker CD73 is known to the skilled person. In this regard CD73refers to cluster of differentiation 73 also known as 5′-nucleotidase(5′-NT) or ecto-5′-nucleotidase. The sequence of the human CD73 proteinmay have the sequence of SEQ ID NO. 1. The marker CD90 is known to theskilled person. In this regard CD90 refers to Cluster of Differentiation90 also known as Thymocyte differentiation antigen 1 (Thy-1). Thesequence of the human CD90 protein may have the sequence of SEQ ID NO:2. The marker CD105 is known to the skilled person. CD105 is also knownas Endoglin (ENG). The sequence of the human CD105 protein may have thesequence of SEQ ID NO: 3.

If a mesenchymal stem cell population of the invention (in particular apopulation of the mesenchymal stem cells of which at least about 98% or99% or express each of the markers CD73, CD90 and CD105 and lackexpression of each of the markers: CD34, CD45 and HLA-DR) is used forclinical trials or as an approved therapeutic, a cell population of theworking cell bank will typically be used for this purpose. As explained,the mesenchymal stem cell population may lack expression of thefollowing markers: CD34, CD45 and HLA-DR. In this context it is notedthat the marker CD34, CD45 and HLA-DR are known to the skilled person.The human CD34 protein may have the sequence of SEQ ID NO. 4. The humanCD45 protein may have the sequence of SEQ ID NO: 5. The human HLA-DRprotein may have the sequence of SEQ ID NO: 6.

Both the stem cell population of the isolation step (which may make upthe master cell bank) and the stem cell population of the subculturingstep (which may make up the working cell bank) can, for example, bestored in cryo-preserved form.

As mentioned above, the present method of isolating mesenchymal stemcells from the amniotic membrane of umbilical cord has the advantagethat all components used in the culture medium of the invention areavailable in GMP quality and thus provide the possibility to isolate themesenchymal stem cells under GMP conditions for subsequent therapeuticadministration.

Thus, the stem cell population can also be an induced pluripotent stemcell population. “Induced pluripotent stem cells”, as used herein, referto adult somatic cells that have been genetically reprogrammed to anembryonic stem cell-like state by being forced to express genes andfactors important for maintaining the defining properties of embryonicstem cells. Thus, induced pluripotent stem cells can bederived/generated from a non-pluripotent cell.

Induced pluripotent stem cells are an important advancement in stem cellresearch, as they allow obtaining pluripotent stem cells without the useof embryos. Mouse iPSCs were first reported in 2006 (Takahashi, K;Yamanaka, S (2006). “Induction of pluripotent stem cells from mouseembryonic and adult fibroblast cultures by defined factors”. Cell 126(4): 663-76), and human iPSCs (hiPSCs) were first reported in 2007(Takahashi et al. (2007) “Induction of pluripotent stem cells from adulthuman fibroblasts by defined factors.” Cell; 131(5):861-72). Mouse iPSCsdemonstrate important characteristics of pluripotent stem cells,including expression of stem cell markers, forming tumors containingcells from all three germ layers, and being able to contribute to manydifferent tissues when injected into mouse embryos at a very early stagein development. Human iPSCs also express stem cell markers and arecapable of generating cells characteristic of all three germ layers.Such stem cell markers can include Oct3/4, Sox2, Nanog, alkalinephosphatase (ALP) as well as stem cell-specific antigen 3 and 4(SSEA3/4). Also, the chromatin methylation patterns of iPSC are similarto that of embryonic stem cells (Tanabe, Takahashi, Yamanaka (2014)“Induction of pluripotency by defined factors.” Proc. Jpn. Acad., 2014,Ser. B 90).

In addition, iPSCs are able to self-renew in vitro and differentiateinto all three germ layers. The pluripotency or the potential todifferentiate into different cell types of iPSC can tested, e.g., by invitro differentiation into neural or glia cells or the production ofgermline chimeric animals through blastocyst injection.

Methods for the generation of human induced pluripotent stem cells arewell known to the skilled person and for example described inWO2009115295, WO2009144008 or EP2218778. Thus, the skilled artisan canobtain an iPSC by any method. In principle, induced pluripotent stemcells may be obtained from any adult somatic cell (of a subject).Exemplary somatic cells include peripheral blood Mononuclear Cells(PBMCs) from blood or fibroblasts obtained from skin tissue biopsies.

The method of the present invention includes that the stem cellpopulation as described herein is contacted with a liquid carrier. It isenvisioned that in the method of the present invention the stem cellpopulation as described herein is contacted with the carrier beforetransporting/storing. Additionally or alternatively, the stem cellpopulation is contacted with the carrier after its harvest. Howharvesting can be performed is described in detail elsewhere herein aswell as in the Experimental Section. For example, the stem cellpopulation can be contacted with the carrier about 0 minutes, about 1minute, about 5 minutes, about 10 minutes, about 30 minutes, about 45minutes, about 60 minutes or a longer time after its harvest.

Harvesting can comprise separating the stem cell population from culturemedium e.g. from PTT-6. Suitable techniques for such separation areknown to the skilled person. For example, separating can be performed bycentrifuging the stem cells within a culture medium and decanting theculture medium.

The stem cell population is contacted with a liquid carrier, wherein theliquid carrier comprises

i) Trolox; ii) Na⁺;

iii) K⁺;

iv) Cl⁻;

v) H₂PO₄ ⁻;

vi) HEPES;

vii) Lactobionate;viii) Sucrose;

ix) Mannitol; x) Glucose; xi) Dextran-40;

xii) Adenosine, andxiii) Glutathione.

By “Trolox” is meant 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylicacid of CAS Number 53188-07-1. It is a water-soluble analog of vitamin Eand is suggested to reduce oxidative stress or damage. FIG. 19 shows thedatasheet of Trolox available from Tocris. It also commerciallyavailable from Sigma Aldrich (product number: 238813).

Both of Na⁺ and Cl⁻ are well known ions. The skilled person knows how toobtain these. For example, these ions may be added to the carrier as aNaCl salt. NaCl in GMP quality can be obtained from Sigma Aldrich. FIG.20 shows the datasheet of NaCl available from Sigma Aldrich.

K⁺ and H₂PO₄ ⁻ (dihydrogen phosphate) are also well known to the skilledperson. It may be used e.g. as a KH₂PO₄ obtainable from SigmaAldrich.FIG. 21 shows the datasheet of KH₂PO₄ available from Sigma Aldrich.

HEPES also named 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (CASNumber 7365-45-9) is commonly used as a zwitterionic organic chemicalbuffering agent. The person skilled in the art also knows where toobtain HEPES, which is commercially available. For example, she/he mayobtain it from Sigma Aldrich; the corresponding data sheet shown in FIG.22 .

Lactobionate is the carboxylate anion of lactobionic acid. Lactobionicacid (4-O-β-galactopyranosyl-D-gluconic acid) is a sugar acid.Lactobionate can be used in different ways. When used as potassiumlactobionate it can e.g. provide osmotic support and prevent cellswelling and when combined with sodium it may have a preservativefunction. Alternatively, mineral salts of lactobionic acid can be usedfor mineral supplementation. For pharmaceutic applications, often theantibiotic erythromycin can inter alia be used as the salt erythromycinlactobionate. The skilled person also knows where to obtain lactobionatee.g. sodium lactobionate (Cas Number: 27297-39-8), namely from e.g.COMBI-BLOCKS, see product sheet in FIG. 23 .

Sucrose, also known as D-Glc-(1→2)-β-D-Fru, α-D-glucopyranosylβ-D-fructofuranoside, β-D-fructofuranosyl-α-D-glucopyranoside,D(+)-saccharose or sugar (CAS Number 57-50-1) can as the othersubstances be commercially obtained and the skilled person knows whereto buy it as well. The corresponding product sheet for sucrose fromSigma Aldrich is shown in FIG. 24 .

Mannitol is a type of sugar alcohol (CAS Registry Number: 69-65-8). Theperson skilled in the art knows how to obtain mannitol. For example, itmay be obtained from Avantor. The respective product sheet is shown inFIG. 25 .

Glucose (CAS Number: 50-99-7) is also well known to the skilled personand commercially available. A respective product sheet from SigmaAldrich is shown in FIG. 26 .

Dextran is a branched glucan composed of linear α (1→6) linked glucoseunits and α (1→3) link initiated branches. Dextran ranges in size from10,000 to 150,000 Kd. Dextrans are used in many applications as volumeextenders, stabilizers, matrix components, binding platforms, lubricantsand physical structure components. Dextran 40 (CAS Number: 9004-54-0) asused in the carrier described herein is typically used in thedevelopment of new improved preservation solutions for organtransplantation. Dextran 40 may be used to determine cell tightness andflux parameters across cell layers, Dextran 40 can also be used as acolloidal plasma volume extender. Dextran-40 is commercially availableand can inter alia be obtained from Sigma Aldrich (product sheet shownin FIG. 27 ).

Adenosine (CAS Number 58-61-7) is a purine nucleoside composed of amolecule of adenine attached to a ribose sugar molecule (ribofuranose)moiety via a β-N₉-glycosidic bond. Adenosine is commercially availableinter alia from Sigma-Aldrich (the corresponding product sheet is shownin FIG. 28 ).

Glutathione is also known as(2S)-2-Amino-4-{[(1R)-1-[(carboxymethyl)carbamoyl]-2-sulfanylethyl]carbamoyl}butanoicacid. This component is commercially available and can inter alia beobtained from Sigma Aldrich (corresponding product sheet shown in FIG.29 ).

In principle any liquid carrier comprising the substances as listed ini)-xiii) above can be used in the method of the present invention. Thecarrier is a liquid carrier. Thus, it is possible that the substances aslisted in i)-xiii) are dissolved in a liquid to form asolution/suspension. The liquid may be any suitable liquid. For example,the liquid can be a culture medium, water, buffer, or the like.

The carrier may additionally comprise further pH buffers, energysubstrates, free radical scavengers, and osmotic/oncotic stabilizers—allknown to the skilled person. Furthermore, the liquid carrier may beserum-free and/or protein-free. The liquid carrier may not comprise adipolar aprotic solvent such as for example DMSO. In particular, theliquid carrier may be a carrier as described in WO 2010/064054. Thecarrier may be HypoThermosol™ or HypoThermosol™-FRS (HTS-FRS).HypoThermosol™-FRS (HTS-FRS) can be purchased from STEMCELL Technologies(according to the respective product sheet shown in FIG. 30 ).

It is further envisioned that the carrier is a transport/storage mediumor an excipient. A transport/storage medium, may be a natural medium,which consists solely of naturally occurring biological fluids, whichadditionally comprise substances as listed in i)-xiii) as describedherein. The medium can also be one comprising substances as listed ini)-xiii) as described herein and addition of (further) nutrients (bothorganic and inorganic), vitamins, salts, O₂ and CO₂ gas phases, serumproteins, carbohydrates, and/or cofactors. In particular embodiments themedium is serum and/or protein free.

The carrier may also be an excipient. An “excipient” is a substanceformulated alongside the active ingredient of a medication. In thepresent method the active ingredient is the stem cell population.

The carrier may further comprise biocompatible scaffolds ormicrocarriers. The scaffolds or microcarriers can, for example, bebiodegradable polymeric substances, most preferably poly(D,Llactic-co-glycolic acid) (PLGA)). Alternatively, the scaffolds ormicro-carriers may be smooth, macroprorous or microporous structurescomprising substances including poly-L-lactide (PLLA), collagen,fibronectin, glycosaminoglycans (GAGs), fibrin, starch, cellulosearabinogalactan (larch gum), alginic acid, agar, carrageenan, chitin,hyaluronic acid, dextran, gellan gum, pullulan, hydroxyapatite,polyhydroxyalkanoates (PHAs), hydrogels or other self-assemblingmaterials such as peptide based nanostructured fibrous scaffolds.

In principle any amount of stem cells can be contacted with any amountof liquid carrier. In this regard the contacting can be performed bysuspending the stem cell population in a density of about 70 million/ml,of about 60 million/ml, of about 50 million/ml, of about 40 million/ml,of about 30 million/ml, of about 20 million/ml, of about 10 million/ml,of about 5 million/ml, of about 4 million/ml, of about 3 million/ml, ofabout 2 million/ml, of about 1 million/ml, of about 0.5 million/ml, ofabout 0.1 million/ml or of less than 0.1 million cells in 1 ml of thecarrier. In some embodiments, the contacting is performed by suspendingthe stem cell population in a density of about 10 million/1 ml carrier.

After contacting the stem cell population with the carrier, the stemcells contacted with the carrier can be aliquoted into vials in a volumeof about 50 ml, of about 20 ml, of about 10 ml, of about 5 ml, of about4 ml, of about 3 ml, of about 2 ml, of about 1 ml, of about 0.5 ml, ofabout 0.25 ml or of less than 0.25 ml carrier. For example, the stemcells that have been contacted with the carrier can be aliquoted intovials in a volume of about 1 ml.

It is further envisioned that the method of the present invention doesnot comprise a thawing or freezing step. This may include that aftertheir harvest the stem cell population is transported/stored without theneed to freeze and thaw the stem cell population.

The carrier used in the method of transporting/storing the stem cellpopulation as described herein is particularly suited for this purpose.One advantage of this carrier is that substantially all stem cellstransported/stored therein remain viable. A “viable cell” is a cell ableto live. The person skilled in the art knows how to detect viable cells.One such method is staining cells with the dye Trypan blue. Viable cellsdo not stain positive with Trypan blue.

In this regard, in the method of the present invention at most about50%, about 40%, about 30%, about 20%, about 10% or less than about 10%of the stem cells of the population may die during transporting/storingcompared to the number/amount of viable stem cells beforetransporting/storing.

The method of the present invention also contemplates that the stem cellpopulation has any cell diameter after transporting/storage. The personskilled in the art knows how to measure the diameter of a cell. Forexample, cell size/diameter may be determined by capturing a microscopeimage and using secondary software to measure the diameter of the cell.Most of the stem cells in the stem cell population can therefore have acell diameter between about 9 μm and about 20 μm aftertransporting/storage. It is also envisioned that most of the stem cellsin the stem cell population have a cell diameter between about 12 μm andabout 16 μm after transporting.

The stem cells transported/stored in the carrier as described hereinsecrete the same proteins/factors as viable stem cells. For example, themethod of the present invention contemplates that aftertransport/storage the (mesenchymal) stem cell population may secreteabout as much TGFbeta 1 as before transporting/storage. TGFbeta 1(Transforming growth factor beta, TGF-β1) is known to the skilled personand may comprise the sequence as shown in SEQ ID NO. 7. Additionally oralternatively, after transporting/storing the (mesenchymal) stem cellpopulation may secrete about as much VEGF (Vascular endothelial growthfactor), PDGF-AA (Platelet-derived growth factor subunit AA), Ang-1(Angiogenin-1), and/or HGF (Hepatocyte growth factor) as beforetransporting/storing. All of VEGF, PDGF-AA, Ang-1, and/or HGF are knownto the skilled person for their involvent in wound healing. Inparticular, VEGF may comprise a sequence as shown in SEQ ID NO. 8,PDGF-AA may have a sequence as shown in SEQ ID NO. 9, Ang-1 may have asequence as shown in SEQ ID NO. 10 while HGF may have a sequence asshown in SEQ ID NO. 11. Additionally or alternatively, essentially noPDGF-BB and/or IL-10 is detected before and/or after transporting. Bothof PDGF-BB (Platelet-derived growth factor subunit BB) and/or IL-10(interleukin-10) are also known to the skilled person. PDGF-BB maycomprise a sequence as shown in SEQ ID NO. 12 while IL-10 may comprise asequence as shown in SEQ ID NO: 13. The secretion of these factors canbe determined with any suitable method, for example, by measuring theamount of protein (i.e., for example, PDGF-AA, PDGF-BB, VEGF, IL-10,Ang-1, HGF or TGFβ1) that the stem cells secrete into the carrier. Theamount of protein can be measured by commercially availableantibodies/immunoassays in an automated fashion, using, for example asystem such as the FLEXMAP 3D system (Luminex Corporation, Austin, Tex.,USA). In this context, it is noted that involvement of the proteinsAngiopoietin 1 (Ang-1), TGF-β1, VEGF, and HGF in the wound healingprocess is known to the person skilled in the art. For the involvementof Angiopoietin 1 in wound healing, see, for example, Li et al. StemCell Research & Therapy 2013, 4:113 “Mesenchymal stem cells modifiedwith angiopoietin-1 gene promote wound healing”. For the involvement ofHepatocyte Growth Factor (HGF) in wound healing, in particular healingof chronic/non healing wounds see for example, Yoshida et al.,“Neutralization of Hepatocyte Growth Factor Leads to RetardedCutaneousWound Healing Associated with Decreased Neovascularization andGranulation Tissue FormationJ. Invest. Dermatol. 120:335-343, 2003, Li,Jin-Feng et al. “HGF Accelerates Wound Healing by Promoting theDedifferentiation of Epidermal Cells through β 1-Integrin/ILK Pathway.”BioMed Research International 2013 (2013): 470418 or Conway et al,“Hepatocyte growth factor regulation: An integral part of why woundsbecome chronic”. Wound Rep Reg (2007) 15 683-692. For the involvement ofVascular Endothelial Growth Factor (VEGF) in wound healing, inparticular healing of chronic/non-healing wounds, see for example Frogetet al., Eur. Cytokine Netw., Vol. 14, March 2003, 60-64 or Bao et al.,“The Role of Vascular Endothelial Growth Factor in Wound Healing” J SurgRes. 2009 May 15; 153(2): 347-358.

For the involvement of Transforming Growth Factor Beta (includingTGF-β1, TGF-β2, and TGF-β3) in wound healing, in particular healing ofchronic/non-healing wounds see for example, Ramirez et al. “The Role ofTGFb Signaling in Wound Epithelialization” Advances In Wound Care,Volume 3, Number 7, 2013, 482-491 or Pakyari et al., Critical Role ofTransforming Growth Factor Beta in Different Phases of Wound Healing,Advances In Wound Care, Volume 2, Number 5, 2012, 215-224.

Turning now to the culture medium used in the present invention, theculture medium may comprise, for the isolation or cultivation of themesenchymal cord lining stem cells, DMEM in a final concentration ofabout 55 to 65% (v/v), F12 in a final concentration of about 5 to 15%(v/v), M171 in a final concentration of about 15 to 30% (v/v) and FBS ina final concentration of about 1 to 8% (v/v). The value of “% (v/v)” asused herein refers to the volume of the individual component relative tothe final volume of the culture medium. This means, if DMEM is, forexample, present in the culture medium at a final concentration of about55 to 65% (v/v), 1 liter of culture medium contains about 550 to 650 mlDMEM.

In other embodiments, the culture medium may comprise DMEM in a finalconcentration of about 57.5 to 62.5% (v/v), F12 in a final concentrationof about 7.5 to 12.5% (v/v), M171 in a final concentration of about 17.5to 25.0% (v/v) and FBS in a final concentration of about 1.75 to 3.5%(v/v). In further embodiments, the culture medium may comprise DMEM in afinal concentration of about 61.8% (v/v), F12 in a final concentrationof about 11.8% (v/v), M171 in a final concentration of about 23.6% (v/v)and FBS in a final concentration of about 2.5% (v/v).

In addition to the above-mentioned components, the culture medium maycomprise supplements that are advantageous for cultivation of themesenchymal cord lining stem cells. The culture medium of the presentinvention may, for example, comprise Epidermal Growth Factor (EGF). Ifpresent, EGF may be present in the culture medium in a finalconcentration of about 1 ng/ml to about 20 ng/ml. In some of theseembodiments, the culture medium may comprise EGF in a finalconcentration of about 10 ng/ml.

The culture medium may also comprise insulin. If present, insulin may bepresent in a final concentration of about 1 μg/ml to 10 μg/ml. In someof these embodiments, the culture medium may comprise Insulin in a finalconcentration of about 5 μg/ml.

The culture medium may further comprise at least one of the followingsupplements: adenine, hydrocortisone, and 3,3′,5-Triiodo-L-thyroninesodium salt (T3). In such embodiments, the culture medium may compriseall three of adenine, hydrocortisone, and 3,3′,5-Triiodo-L-thyroninesodium salt (T3). In these embodiments, the culture medium may compriseadenine in a final concentration of about 0.05 to about 0.1 μg/ml,hydrocortisone in a final concentration of about 1 to about 10 μg/mland/or 3,3′,5-Triiodo-L-thyronine sodium salt (T3) in a finalconcentration of about 0.5 to about 5 ng/ml.

In one embodiment, the mesenchymal stem cells are cultured in PTT6medium to obtain the highly purified mesenchymal stem cell populationdescribed and used herein. In this context it is noted that PTT6 mediumas described herein is obtained by mixing to obtain a final volume of500 ml culture medium:

i. 250 ml of DMEM

ii. 118 ml M171

iii. 118 ml DMEM/F12

iv. 12.5 ml Fetal Bovine Serum (FBS) to reach a final concentration of2.5% (v/v)

v. EGF in a final concentration of 10 ng/ml

vi. Insulin in a final concentration of 5 μg/ml.

vii. Insulin 0.175 ml (final concentration of 5 μg/ml)

By “DMEM” is meant Dulbecco's modified eagle medium which was developedin 1969 and is a modification of basal medium eagle (BME) (cf. FIG. 1showing the data sheet of DMEM available from Lonza). The original DMEMformula contains 1000 mg/L of glucose and was first reported forculturing embryonic mouse cells. DMEM has since then become a standardmedium for cell culture that is commercially available from varioussources such as ThermoFisher Scientific (catalogue number 11965-084),Sigma Aldrich (catalogue number D5546) or Lonza, to new only a fewsuppliers. Thus, any commercially available DMEM can be used in thepresent invention. In preferred embodiments, the DMEM used herein is theDMEM medium available from Lonza under catalog number 12-604F. Thismedium is DMEM supplemented with 4.5 g/L glucose and L-glutamine. Inanother preferred embodiment the DMEM used herein is the DMEM medium ofSigma Aldrich catalogue number D5546 that contains 1000 mg/L glucose,and sodium bicarbonate but is without L-glutamine.

By “F12” medium is meant Ham's F12 medium. This medium is also astandard cell culture medium and is a nutrient mixture initiallydesigned to cultivate a wide variety of mammalian and hybridoma cellswhen used with serum in combination with hormones and transferrin (cf.FIG. 2 , showing the data sheet of Ham's F12 medium from Lonza). Anycommercially available Ham's F12 medium (for example, from ThermoFisherScientific (catalogue number 11765-054), Sigma Aldrich (catalogue numberN4888) or Lonza, to name only a few suppliers) can be used in thepresent invention. In preferred embodiments, Ham's F12 medium from Lonzais used.

By “DMEM/F12” or “DMEM:F12” is meant a 1:1 mixture of DMEM with Ham'sF12 culture medium (cf. FIG. 3 showing the data sheet for DMEM: F12(1:1) medium from Lonza). DMEM/F12 (1:1) medium is a widely used basalmedium for supporting the growth of many different mammalian cells andis commercially available from various suppliers such as ThermoFisherScientific (catalogue number 11330057), Sigma Aldrich (catalogue numberD6421) or Lonza. Any commercially available DMEM:F12 medium can be usedin the present invention. In preferred embodiments, the DMEM:F12 mediumused herein is the DMEM/F12 (1:1) medium available from Lonza undercatalog number 12-719F (which is DMEM: F12 with L-glutamine, 15 mMHEPES, and 3.151 g/L glucose).

By “M171” is meant culture medium 171, which has been developed as basalmedium for the culture and growth of normal human mammary epithelialcells (cf. FIG. 4 showing the data sheet for M171 medium from LifeTechnologies Corporation). This basal medium is widely used and iscommercially available from suppliers such as ThermoFisher Scientific orLife Technologies Corporation (catalogue number M171500), for example.Any commercially available M171 medium can be used in the presentinvention. In preferred embodiments, the M171 medium used herein is theM171 medium available from Life Technologies Corporation under cataloguenumber M171500.

By “FBS” is meant fetal bovine serum (that is also referred to as “fetalcalf serum”), i.e. the blood fraction that remains after the naturalcoagulation of blood, followed by centrifugation to remove any remainingred blood cells. Fetal bovine serum is the most widely usedserum-supplement for in vitro cell culture of eukaryotic cells becauseit has very low level of antibodies and contains more growth factors,allowing for versatility in many different cell culture applications.The FBS is preferably obtained from a member of the International SerumIndustry Association (ISIA) whose primary focus is the safety and safeuse of serum and animal derived products through proper origintraceability, truth in labeling, and appropriate standardization andoversight. Suppliers of FBS that are ISIA members include AbattoirBasics Company, Animal Technologies Inc., Biomin Biotechnologia LTDA, GEHealthcare, Gibco by Thermo Fisher Scientific and Life ScienceProduction, to mention only a few. In currently preferred embodiments,the FBS is obtained from GE Healthcare under catalogue number A15-151.

As mentioned above, a method of making a culture medium for isolatingthe mesenchymal stem cell population used in the invention comprisesmixing to obtain a final volume of 500 ml culture medium:

i. 250 ml of DMEM

ii. 118 ml M171

iii. 118 ml DMEM/F12

iv. 12.5 ml Fetal Bovine Serum (FBS) to reach a final concentration of2.5% (v/v).

As explained above, DMEM/F12 medium is a 1:1 mixture of DMEM and Ham'sF12 medium. Thus, 118 ml DMEM/F12 medium contain 59 ml DMEM and 59 mlF12. Accordingly, when using this method of making a culture medium, thefinal concentrations (v/v) with 500 ml total volume are as follows:

DMEM: 250 ml+59 ml=309 ml, corresponds to 309/500=61.8% (v/v)

M171: 118 ml, corresponds to 118/500=23.6% (v/v)

F12: 59 ml, corresponds to 59/500=11.8% (v/v).

Embodiments of this method of making a culture medium further compriseadding

v. 1 ml EGF stock solution (5 μg/ml) to achieve a final EGFconcentration of 10 ng/ml, and

vi. Insulin 0.175 ml stock solution (14.28 mg/ml) to achieve a finalinsulin concentration of 5 μg/ml.

It is noted here that in these embodiments, the above-mentioned volumesof these components i. to vi. will result in a final volume of 499.675ml culture medium. If no further components are added to the culturemedium, the remaining 0.325 ml (to add up to a volume of 500 ml) can,for example, be any of components i. to iv., that means either DMEM,M171, DMEM/F12 or FBS. Alternatively, the concentration of the stocksolution of EGF or Insulin can of course be adjusted such that the totalvolume of the culture medium is 500 ml. In addition, it is also notedthat components i. to iv. do not necessarily have to be added in theorder in which they are listed but it is of course also possible to useany order to mix these components to arrive at the culture medium of thepresent invention. This means, that for example, M171 and DMEM/F12 canbe mixed together and then combined with DMEM and FBS to reach finalconcentrations as described here, i.e. a final concentration of DMEM ofabout 55 to 65% (v/v), a final concentration of F12 of about 5 to 15%(v/v), a final concentration of M171 of about 15 to 30% (v/v) and afinal concentration of FBS of about 1 to 8% (v/v).

In other embodiments, the method further comprises adding to DMEM avolume of 0.325 ml of one or more of the following supplements: adenine,hydrocortisone, 3,3′,5-Triiodo-L-thyronine sodium salt (T3), therebyreaching a total volume of 500 ml culture medium. In this embodiment,the final concentration of these supplements in DMEM may be as follows:

about 0.05 to 0.1 μg/ml adenine, for example about 0.025 μg/ml adenine,about 1 to 10 μg/ml hydrocortisone,about 0.5 to 5 ng/ml 3,3′,5-Triiodo-L-thyronine sodium salt (T3), forexample 1.36 ng/ml 3,3′,5-Triiodo-L-thyronine sodium salt (T3).

In line with the above disclosure, a cell culture medium used herein isobtainable or that is obtained by the method of making the medium asdescribed here.

In addition, a method of isolating mesenchymal stem cells from theamniotic membrane of the umbilical cord, wherein this method comprisescultivating amniotic membrane tissue in the culture medium prepared bythe method is described here.

Thus, the present invention is also directed to (the use of) a cellculture medium comprising:

-   -   DMEM in the final concentration of about 55 to 65% (v/v),    -   F12 in a final concentration of about 5 to 15% (v/v),    -   M171 in a final concentration of about 15 to 30% (v/v) and    -   FBS in a final concentration of about 1 to 8% (v/v).

In certain embodiments of the culture medium described here, the mediumcomprises DMEM in the final concentration of about 57.5 to 62.5% (v/v),F12 in a final concentration of about 7.5 to 12.5% (v/v), M171 in afinal concentration of about 17.5 to 25.0% (v/v) and FBS in a finalconcentration of about 1.75 to 3.5% (v/v). In other embodiments theculture medium may comprise DMEM in a final concentration of about 61.8%(v/v), F12 in a final concentration of about 11.8% (v/v), M171 in afinal concentration of about 23.6% (v/v) and FBS in a finalconcentration of about 2.5% (v/v).

In addition, the culture medium may further comprise Epidermal GrowthFactor (EGF) in a final concentration of about 1 ng/ml to about 20ng/ml. In certain embodiments, the culture medium comprises EGF in afinal concentration of about 10 ng/ml. The culture medium describedherein may further comprise Insulin in a final concentration of about 1μg/ml to 10 μg/ml. In such embodiments the culture medium may compriseInsulin in a final concentration of about 5 μg/ml.

The cell culture medium may further comprise at least one of thefollowing supplements: adenine, hydrocortisone, and3,3′,5-Triiodo-L-thyronine sodium salt (T3). In certain embodiments theculture medium comprises all three of adenine, hydrocortisone, and3,3′,5-Triiodo-L-thyronine sodium salt (T3). If present, the culturemedium may comprise adenine in a final concentration of about 0.01 toabout 0.1 μg/ml adenine or of about 0.05 to about 0.1 μg/ml adenine,hydrocortisone in a final concentration of about 0.1 to about 10 μg/mlhydrocortisone or of about 1 to about 10 μg/ml hydrocortisone and/or3,3′,5-Triiodo-L-thyronine sodium salt (T3) in a final concentration ofabout 0.5 to about 5 ng/ml.

In embodiments of the cell culture medium, 500 ml of the cell culturemedium of the present invention comprise:

i. 250 ml of DMEM ii. 118 ml M171 iii. 118 ml DMEM/F12

iv. 12.5 ml Fetal Bovine Serum (FBS) (final concentration of 2.5%)In further embodiments, the cell culture medium may further comprisev. EGF in a final concentration of 10 ng/ml, andvi. Insulin in a final concentration of 5 μg/ml.Both, insulin and and EGF can be added to to the culture medium using astock solution of choice, such that the total volume of the culturemedium does not exceed 500 ml.

In a particular example, the components i. to vi. of the culture mediumused in the present invention are the components indicated in FIG. 5 ,meaning they are obtained from the respective manufacturers using thecatalogue number indicated in FIG. 5 . The medium that is obtained frommixing the components i. to vi. as indicated in FIG. 5 is also referredherein as “PTT-6”. It is again noted in this context that theconstituents i. to vi. as well as any other ingredient such as anantibiotic of any other commercial supplier can be used in making themedium of the present invention.

In addition, the cell culture medium of the invention may compriseadenine in a final concentration of about 0.01 to about 0.1 μg/mladenine or of about 0.05 to about 0.1 μg/ml adenine, hydrocortisone in afinal concentration of about 0.1 to 10 μg/ml, of about 0.5 to about 10μg/ml, or of about 1 to about 10 μg/ml hydrocortisone and/or3,3′,5-Triiodo-L-thyronine sodium salt (T3) in a final concentration ofabout 0.1 to about 5 ng/ml or of about 0.5 to about 5 ng/ml.

To obtain the mesenchymal stem cell population as described herein theumbilical cord tissue may be cultured till a suitable number of(primary) mesenchymal cord lining stem cells have outgrown from thetissue. In typical embodiments, the umbilical cord tissue is cultivateduntil cell outgrowth of the mesenchymal stem cells of the amnioticmembrane reaches about 70 to about 80% confluency. It is noted here thatthe term “confluency” or “confluence” is used in its regular meaning inthe art of cell culture and is meant as an estimate/indicator of thenumber of adherent cells in a culture dish or a flask, referring to theproportion of the surface which is covered by cells. For example, 50percent confluence means roughly half of the surface is covered andthere is still room for cells to grow. 100 percent confluence means thesurface is completely covered by the cells, and no more room is left forthe cells to grow as a monolayer.

Once a suitable number of primary cells (mesenchymal cord lining stemcells) have been obtained from the cord lining tissue by tissue explant,the mesenchymal stem cells are removed from the cultivation containerused for the cultivation. By so doing, a master cell bank containing the(primary) isolated mesenchymal stem cells of the amniotic membrane canbe obtained. Typically, since mesenchymal stem cells are adherent cells,removing is carried out using standard enzymatic treatment. For example,the enzymatic treatment may comprise trypsination as described inInternational US patent application 2006/0078993, International patentapplication WO2006/019357 or International patent applicationWO2007/046775, meaning outgrowing cells can be harvested bytrypsinization (0.125% trypsin/0.05% EDTA) for further expansion. If theharvested mesenchymal stem cells are, for example, used for generating amaster cell bank, the cells can also be cryo-preserved and stored forfurther use as explained herein below.

Once being harvested, the mesenchymal stem cells can be transferred to acultivation container for subculturing. The subculturing can also bestarted from frozen primary cells, i.e. from the master cell bank. Forsubculturing, any suitable amount of cells can be seeded in acultivation container such as cell culture plate. The mesenchymal stemcells can, for this purpose, be suspended in a suitable medium (mostconveniently, the culture medium PTT-6) for subculturing at aconcentration of, for example, about 0.5×10⁶ cells/ml to about 5.0×10⁶cells/ml. In one embodiment the cells are suspended for subcultivationat a concentration of about 1.0×10⁶ cells/ml. The subculturing can becarried out by cultivation either in simple culture flasks but also, forexample, in a multilayer system such as CellStacks (Corning, Corning,N.Y., USA) or Cellfactory (Nunc, part of Thermo Fisher Scientific Inc.,Waltham, Mass., USA) that can be stacked in incubators. Alternatively,the subculturing can also be carried out in a closed self-containedsystem such as a bioreactor. Different designs of bioreactors are knownto the person skilled in the art, for example, parallel-plate,hollow-fiber, or micro-fluidic bioreactors. See, for example, Sensebe etal. “Production of mesenchymal stromal/stem cells according to goodmanufacturing practices: a review”, supra. An illustrative example of acommercially available hollow-fiber bioreactor is the Quantum® CellExpansion System (Terumo BCT, Inc). that has, for example, been used forthe expansion of bone marrow mesenchymal stem cells for clinical trials(cf., Hanley et al, Efficient Manufacturing of Therapeutic MesenchymalStromal Cells Using the Quantum Cell Expansion System, Cytotherapy. 2014August; 16(8): 1048-1058). Another example of commercially availablebioreactors that can be used for the subculturing of the mesenchymalstem cell population of the present invention is the Xuri Cell ExpansionSystem available from GE Heathcare. The cultivation of the mesenchymalstem cell population in an automated system such as the Quantum® CellExpansion System is of particular benefit if a working cell bank fortherapeutic application is to be produced under GMP conditions and ahigh number of cells is wanted.

The subculturing of the mesenchymal cord ling stem cells describedherein takes place in a culture medium described herein such as thePTT-6 medium. Accordingly, the culture medium such as PTT-6 can be usedboth for the isolation of the mesenchymal stem cells from the amnioticmembrane and the subsequent cultivation of the isolated primary cells bysubcultivation. Also for the subcultivation, the mesenchymal stem cellscan be cultured till a suitable number of cells have grown. Inillustrative embodiments the mesenchymal stem cells are subcultured tillthe mesenchymal stem cells reach about 70 to about 80% confluency.

The isolation/cultivation of the population of mesenchymal cord liningstem cells can be carried out under standard conditions for thecultivation of mammalian cells. Typically, the method of the inventionof isolating the population of the mesenchymal cord lining stem cells istypically carried out at conditions (temperature, atmosphere) that arenormally used for cultivation of cells of the species of which the cellsare derived. For example, human umbilical cord tissue and themesenchymal cord lining stem cells, respectively, are usually cultivatedat 37° C. in air atmosphere with 5% CO₂. In this context, it is notedthat the mesenchymal cells may be derived of any mammalian species, suchas mouse, rat, guinea pig, rabbit, goat, horse, dog, cat, sheep, monkeyor human, with mesenchymal stem cells of human origin being preferred inone embodiment.

Once a desired/suitable number of mesenchymal cord lining stem cellshave been obtained from the subculture, the mesenchymal stem cells canbe harvested by removing them from the cultivation container used forthe subcultivation. The harvesting of the mesenchymal stem cells istypically again carried out by enzymatic treatment, includingtrypsination of the cells. The isolated mesenchymal stem cells aresubsequently collected and are either directly used or preserved forfurther use. Typically, preserving is carried out by cryo-preservation.The term “cryo-preservation” is used herein in its regular meaning todescribe a process where the mesenchymal stem cells are preserved bycooling to low sub-zero temperatures, such as (typically) −80° C. or−196° C. (the boiling point of liquid nitrogen). Cryo-preservation canbe carried out as known to the person skilled in the art and can includethe use of cryo-protectors such as dimethylsulfoxide (DMSO) or glycerol,which slow down the formation of ice-crystals in the cells of theumbilical cord.

The isolated population of the mesenchymal cord lining stem cells thatis obtained by the isolation method as described herein is highlydefined and homogenous. In typical embodiments of the method at leastabout 90% or more, about 91% or more, about 92% or more, about 93% ormore, about 94% or more, about 95% or more, about 96% or more, about 97%or more, about 98% or more about 99% or more of the isolated mesenchymalstem cells express the following markers: CD73, CD90 and CD105. Inaddition, in these embodiments at least about 90% or more, about 91% ormore, about 92% or more, about 93% or more, about 94% or more, about 95%or more, about 96% or more, about 97% or more, about 98% or more about99% or more of the isolated mesenchymal stem cells may lack expressionof the following markers: CD34, CD45 and HLA-DR. In particularembodiments, about 97% or more, about 98% or more, or about 99% or moreof the isolated mesenchymal stem cell population express CD73, CD90 andCD105 while lacking expression of CD34, CD45 and HLA-DR.

Thus, in line with the above disclosure a mesenchymal stem populationisolated from the amniotic membrane of the umbilical cord, wherein atleast about 90% or more cells of the stem cell population express eachof the following markers: CD73, CD90 and CD105. In preferred embodimentsat least about 91% or more, about 92% or more, about 93% or more, about94% or more, about 95% or more, about 96% or more, about 97% or more,about 98% or more about 99% or more cells of the isolated mesenchymalstem cell population are CD73+, CD90+ and CD105+, meaning that thispercentage of the isolate cell population express each of CD73, CD90 andCD105 (cf. the Experimental Section of the present application) can beused herein. In addition, at least about 90% or more, about 91% or more,about 92% or more, about 93% or more, about 94% or more, about 95% ormore, about 96% or more, about 97% or more, about 98% or more about 99%or more of the isolated mesenchymal stem cells may lack expression ofthe lack expression of the following markers. In particular embodimentsabout 97% or more, about 98% or more, or about 99% or more cells of theisolated mesenchymal stem cell population express CD73, CD90 and CD105while lacking expressing of CD34, CD45 and HLA-DR. Such a highlyhomogenous population of mesenchymal stem cells derived from theamniotic membrane of the umbilical cord has been reported for the firsttime in U.S. provisional application No. 62/404,582, filed Oct. 5, 2016as well as in co-pending U.S. application Ser. No. 15/725,913, filed 5Oct. 2017 as well as in co-pending PCT application PCT/SG2017/050500,also filed 5 Oct. 2017, and meets the criteria for mesenchymal stemcells to be used for cellular therapy (also cf. the Experimental Sectionand, for example, Sensebe et al. “Production of mesenchymal stromal/stemcells according to good manufacturing practices: a review”, supra). Itis noted in this context that this mesenchymal stem cell population canbe obtained by either the isolating method of the present invention butalso by a different method such as cell sorting, if needed.

A method of making a culture medium for isolating mesenchymal stem cellsas described herein can comprise, mixing to obtain a final volume of 500ml culture medium:

i. 250 ml of DMEM ii. 118 ml M171 iii. 118 ml DMEM/F12

iv. 12.5 ml Fetal Bovine Serum (FBS) to reach a final concentration of2.5% (v/v).

As explained above, DMEM/F12 medium is a 1:1 mixture of DMEM and Ham'sF12 medium.

Thus, 118 ml DMEM/F12 medium contain 59 ml DMEM and 59 ml F12.Accordingly, when using this method of making a culture medium, thefinal concentrations (v/v) with 500 ml total volume are as follows:

DMEM: 250 ml+59 ml=309 ml, corresponds to 309/500=61.8% (v/v)M171: 118 ml, corresponds to 118/500=23.6% (v/v)F12: 59 ml, corresponds to 59/500=11.8% (v/v).

The present invention also relates to a method of treating a subjecthaving a disease, the method comprising topically administering amesenchymal stem cell population as described herein to the subject,wherein the mesenchymal stem cell population is administered withinabout 96 hours from the time point the mesenchymal stem cell populationhas been harvested.

Similarly, the present invention also relates to mesenchymal stem cellpopulation as described herein for use in a method of treating a diseaseof a subject, wherein the mesenchymal stem cell population is topicallyadministered within about 96 hours from the time point the mesenchymalstem cell population has been harvested

The subject to be treated may be any suitable subject. The subject canbe a vertebrate, more preferably a mammal. Mammals include, but are notlimited to, farm animals, sport animals, pets, primates, dogs, horses,mice and rats. A mammal can also be a human, dog, cat, cow, pig, mouse,rat etc. Thus, in one embodiment, the subject is a vertebrate. Thesubject can also be a human subject. The subject therefore can be asubject in need of treatment. As such the subject may be afflicted witha disease as described elsewhere herein. In some embodiments the subjectis afflicted with Type I or Type II diabetes with chronic foot ulcers.Preferably, the subject is negative for HLA antibodies to themesenchymal stem cell population.

The mesenchymal stem cell population may be applied in any dosage. Thedosage may be therapeutically effective. The “therapeutically effectiveamount/dosage” can vary with factors including but not limited to theactivity of the cells used, stability of the cells in the patient'sbody, the severity of the conditions to be alleviated, the age andsensitivity of the patient to be treated, adverse events, and the like,as will be apparent to a skilled artisan. The amount of administrationcan be adjusted as the various factors change over time.

The dosage in which the mesenchymal stem cells are applied can also be aunit dosage. For example, the mesenchymal stem cell population can beapplied in a unit dosage of about 20 million cells, of about 15 millioncells, of about 10 million cells, of about 5 million cells, of about 4million cells, of about 3 million cells, of about 2 million cells, ofabout 1 million cells, of about 0.5 million cells, of about 0.25 millioncells or of less than 0.25 million cells. In a particular embodiment,the mesenchymal stem cell population is applied in a unit dosage ofabout 10 million cells.

The mesenchymal stem cells may be applied several times to the samesubject. For example, stem cells are applied once, twice, three times ormore a week. In principle any unit dosage of mesenchymal stem cells maybe applied for the number of times suitable to cure or alleviate thedisease. For example, the mesenchymal stem cell population can beapplied once, twice three times or more a week. The mesenchymal stemcell population may also be applied for one, two, three, four, five,six, seven, eight, nine, ten, elven weeks or more.

Thus, the unit dosage of about 20 million cells, of about 15 millioncells, of about 10 million cells, of about 5 million cells, of about 4million cells, of about 3 million cells, of about 2 million cells, ofabout 1 million cells, of about 0.5 million cells, of about 0.25 millioncells or of less than 0.25 million cells is administered once or twice aweek. The unit dosage of about 20 million cells, of about 15 millioncells, of about 10 million cells, of about 5 million cells, of about 4million cells, of about 3 million cells, of about 2 million cells, ofabout 1 million cells, of about 0.5 million cells, of about 0.25 millioncells or of less than 0.25 million cells can also be administered one ortwice a week for a period of time of three weeks, of four weeks, or fiveweeks or of six weeks, or of seven weeks, or of eight weeks or of tenweeks or more weeks.

It is also contemplated by the method of treatment of the presentinvention that the mesenchymal stem cell population is applied in adosage of about 1000 cells/cm² to about 5 million cells/cm². Here, theexpression cm² means the area of the wound/skin to which the stem cellsare applied. It is also envisioned that the mesenchymal stem cellpopulation is applied in a dosage of about 100,000 cells/cm², 300,000cells/cm² or 500,000 cells/cm². The mesenchymal stem cell population canalso be applied two times a week for about 8 weeks in a dosage of about100,000 cells/cm², about 300,000 cells/cm² or about 500,000 cells/cm².

The mesenchymal stem cell population is administered within about 96hours from the time point where the mesenchymal stem cell population hasbeen harvested. How harvesting can take place is described elsewhereherein. It is also possible that the mesenchymal stem cell population isapplied within about 72 hours, about 48 hours, about 24 hours, about 12hours, about 6 hours or less from the time point where the mesenchymalstem cell population has been harvested. Between the time of harvestingand application, the mesenchymal stem cell population may be transportedor stored by the method of transporting/storing of the presentinvention. Thus, aspects as described for the method oftransporting/storing of the present application equally relate to themethod of treatment of the present invention mutatis mutandis.

The method of treatment of the present invention serves to alleviate adisease suffered by the subject. In principle, any disease that may betreated by the mesenchymal stem cell population as described herein ismeant here. In particular, the disease may be a skin disease or a wound.The wound may be caused by any cause e.g. by a burn, a bite, a trauma, asurgery, or a disease. The wound can also be caused by diabetic disease.Therefore, the wound can also be a diabetic wound. The wound may also bea diabetic foot ulcer. Notably, the mesenchymal stem cell populationmay, for example, be placed directly onto a wound such as a burn or adiabetic wound (see International patent application WO2007/046775).

As described herein, between the harvesting of the mesenchymal stem cellpopulation as described herein and their application to a subject thecells may be transported/stored in the carrier as defined herein.Therefore, the method of treating a subject of the present invention mayalso comprise the step of separating the mesenchymal stem cellpopulation from the carrier before administering the mesenchymal stemcell population to the subject. The person skilled in the art knows howto perform the separation of cells from a carrier. For example, theseparating of the mesenchymal stem cell population from the carrier maycomprise centrifugation. Additionally or alternatively, separating themesenchymal stem cell population from the carrier can comprisewithdrawing the cell population from the vial by means of syringe.

After separating the stem cells from the carrier or after harvesting themesenchymal stem cells or after obtaining mesenchymal stem cellpopulation as described herein by any other method these cells aretopically applied to a subject. In principle any way of topicaladministration is meant herein. The administering the mesenchymal stemcell population may be performed by means of a syringe. It is howeveralso possible, to contact the mesenchymal stem cells within a cream,ointment, gel, suspension or any other suitable substance beforeapplying the mesenchymal stem cells to the subject. The mesenchymal stemcell population after application to the subject may be held in placee.g. by a dressing such as Tegaderm® dressing and a crepe bandage tocover the Tegaderm® dressing. For a more even distribution of cells theapplication site may be gently massaged.

The present invention also relates to a unit dosage comprising about 20million cells, of about 15 million cells, of about 10 million cells, ofabout 5 million cells, of about 4 million cells, of about 3 millioncells, of about 2 million cells, of about 1 million cells, of about 0.5million cells, of about 0.25 million cells or of less than 0.25 millioncells of a mesenchymal stem cell population as described herein.

It is also envisioned that the unit dosage comprises about 10, about 9,about 8, about 7, about 6, about 5, about 4, about 3, about 2, about 1,about 0.5, about 0.25, or about 0.1 million cells. Preferably the unitdosage comprises about 10 million cells. It is further envisioned thatthe unit dosage comprises about 1000 cells to about 5 million cells. Theunit dosage can be applied in a dosage of about 100,000 cells, 300,000cells or 500,000 cells. As described herein the unit dosage may beapplied topically. For example, the unit dosage may be applied topicallyper cm².

The unit dosage can be applied once, twice, three times or more a week.For example, the unit dosage can be applied for one, two, three, four,five, six, seven, eight, nine, ten, elven weeks or more. The unit dosagecomprising of about 100,000 cells, about 300,000 cells or about 500,000cells can be applied two times a week for 8 weeks, preferably onto 1cm².

The unit dosage can be contained in any suitable container. For example,the unit dosage can be contained in a 1 ml vial. In such cases, forexample 0.1 ml of the vial can be applied onto the subject, preferablyper cm². The unit dosage may alternatively be contained in a syringe.

The unit dosage of the present invention the cells can be in contactwith a liquid carrier as defined herein. If this is the case then themesenchymal stem cells are separated from the carrier beforeadministration. For example, the cells can be centrifuged and isolatedbefore administration to a subject. The carrier may be any carrier asdescribed herein, such as HypoThermosol™ or Hypothermosol™-FRS

The method of treatment and the unit dosage of the present invention cancomprise utilization of viable cells. How viability can be tested isdescribed elsewhere herein.

The invention will be further illustrated by the following non-limitingExperimental Examples.

Sequences as used herein are depicted in below Table 1.

TABLE 1 Sequences as used herein. SEQ ID NO. What Sequence  1 CD73MCPRAARAPATLLLALGAVLWPAAGAWELTILHTNDVHSRLEQTSEDS identifierSKCVNASRCMGGVARLFTKVQQIRRAEPNVLLLDAGDQYQGTIWFTVY P21589 ofKGAEVAHFMNALRYDAMALGNHEFDNGVEGLIEPLLKEAKFPILSANIK Uniprot,AKGPLASQISGLYLPYKVLPVGDEVVGIVGYTSKETPFLSNPGTNLVFED versionEITALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHS number 1 as ofNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIE May 1, 1991:FDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHTDEMFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKFSTGSHCHGSFSLIFLSLWAVIFVLYQ  2 CD90MNLAISIALLLTVLQVSRGQKVTSLTACLVDQSLRLDCRHENTSSSPIQY identifierEFSLTRETKKHVLFGTVGVPEHTYRSRTNFTSKYNMKVLYLSAFTSKDE P04216 ofGTYTCALHHSGHSPPISSQNVTVLRDKLVKCEGISLLAQNTSWLLLLLLS Uniprot, LSLLQATDFMSLversion number 2 as of May 2, 2002:  3 CD 105MDRGTLPLAVALLLASCSLSPTSLAETVHCDLQPVGPERGEVTYTTSQVS identifierKGCVAQAPNAILEVHVLFLEFPTGPSQLELTLQASKQNGTWPREVLLVL P17813ofSVNSSVFLHLQALGIPLHLAYNSSLVTFQEPPGVNTTELPSFPKTQILEWA Uniprot,AERGPITSAAELNDPQSILLRLGQAQGSLSFCMLEASQDMGRTLEWRPRT versionPALVRGCHLEGVAGHKEAHILRVLPGHSAGPRTVTVKVELSCAPGDLDA number 2 as ofVLILQGPPYVSWLIDANHNMQIWTTGEYSFKIFPEKNIRGFKLPDTPQGL Jul. 15, 1998:LGEARMLNASIVASFVELPLASIVSLHASSCGGRLQTSPAPIQTTPPKDTCSPELLMSLIQTKCADDAMTLVLKKELVAHLKCTITGLTFWDPSCEAEDRGDKFVLRSAYSSCGMQVSASMISNEAVVNILSSSSPQRKKVHCLNMDSLSFQLGLYLSPHFLQASNTIEPGQQSFVQVRVSPSVSEFLLQLDSCHLDLGPEGGTVELIQGRAAKGNCVSLLSPSPEGDPRFSFLLHFYTVPIPKTGTLSCTVALRPKTGSQDQEVHRTVFMRLNIISPDLSGCTSKGLVLPAVLGITFGAFLIGALLTAALWYIYSHTRSPSKREPVVAVAAPASSESSSTNHSIGSTQSTP CSTSSMA  4 CD34MLVRRGARAGPRMPRGWTALCLLSLLPSGFMSLDNNGTATPELPTQGT identifierFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITETTVKFTSTSVIT P28906 ofSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTSTS Uniprot,LATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCAEFKK versionDRGEGLARVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEI number 2 as ofSSKLQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKTLIALVTSGALLA Jul. 15, 1998:VLGITGYFLMNRRSWSPTGERLGEDPYYTENGGGQGYSSGPGTSPEAQGKASVNRGAQENGTGQATSRNGHSARQHVVADTEL  5 CD45MYLWLKLLAFGFAFLDTEVFVTGQSPTPSPTGLTTAKMPSVPLSSDPLPT identifierHTTAFSPASTFERENDFSETTTSLSPDNTSTQVSPDSLDNASAFNTTGVSS P08575 ofVQTPHLPTHADSQTPSAGTDTQTFSGSAANAKLNPTPGSNAISDVPGERS Uniprot,TASTFPTDPVSPLTTTLSLAHHSSAALPARTSNTTITANTSDAYLNASETT versionTLSPSGSAVISTTTIATTPSKPTCDEKYANITVDYLYNKETKLFTAKLNVN number 2 as ofENVECGNNTCTNNEVHNLTECKNASVSISHNSCTAPDKTLILDVPPGVEK Jul. 19, 2003:FQLHDCTQVEKADTTICLKWKNIETFTCDTQNITYRFQCGNMIFDNKEIKLENLEPEHEYKCDSEILYNNHKFTNASKIIKTDFGSPGEPQIIFCRSEAAHQGVITWNPPQRSFHNFTLCYIKETEKDCLNLDKNLIKYDLQNLKPYTKYVLSLHAYIIAKVQRNGSAAMCHFTTKSAPPSQVWNMTVSMTSDNSMHVKCRPPRDRNGPHERYHLEVEAGNTLVRNESHKNCDFRVKDLQYSTDYTFKAYFHNGDYPGEPFILHHSTSYNSKALIAFLAFLIIVTSIALLVVLYKIYDLHKKRSCNLDEQQELVERDDEKQLMNVEPIHADILLETYKRKIADEGRLFLAEFQSIPRVFSKFPIKEARKPFNQNKNRYVDILPYDYNRVELSEINGDAGSNYINASYIDGFKEPRKYIAAQGPRDETVDDFWRMIWEQKATVIVMVTRCEEGNRNKCAEYWPSMEEGTRAFGDVVVKINQHKRCPDYIIQKLNIVNKKEKATGREVTHIQFTSWPDHGVPEDPHLLLKLRRRVNAFSNFFSGPIVVHCSAGVGRTGTYIGIDAMLEGLEAENKVDVYGYVVKLRRQRCLMVQVEAQYILIHQALVEYNQFGETEVNLSELHPYLHNMKKRDPPSEPSPLEAEFQRLPSYRSWRTQHIGNQEENKSKNRNSNVIPYDYNRVPLKHELEMSKESEHDSDESSDDDSDSEEPSKYINASFIMSYWKPEVMIAAQGPLKETIGDFWQMIFQRKVKVIVMLTELKHGDQEICAQYWGEGKQTYGDIEVDLKDTDKSSTYTLRVFELRHSKRKDSRTVYQYQYTNWSVEQLPAEPKELISMIQVVKQKLPQKNSSEGNKHHKSTPLLIHCRDGSQQTGIFCALLNLLESAETEEVVDIFQVVKALRKARPGMVSTFEQYQFLYDVIASTYPAQNGQVKKNNHQEDKIEFDNEVDKVKQDANCVNPLGAPEKLPEAKEQAEGSEPTSGTEGPEH SVNGPASPALNQGS  6HLA-DR MAISGVPVLGFFIIAVLMSAQESWAIKEEHVIIQAEFYLNPDQSGEFMFDF identifierDGDEIFHVDMAKKETVWRLEEFGRFASFEAQG ALANIA VDKANLEIMT P01903 ofKRSNYTPITNVPPEVTVLTNSPVELREPNVLICFIDKFTPPVVNVTWLRNG Uniprot,KPVTTGVSETVFLPREDHLFRKFHYLPFLPSTEDVYDCRVEHWGLDEPLL versionKHWEFDAPSPLPETTENVVCALGLTVGLVGIIIGTIFIIKGVRKSNAAERR number 1 as of GPLJul. 21, 1986:  7 Human MEAAVAAPRPRLLLLVLAAAAAAAAALLPGATALQCFCHLCTKDNFTCTGFbetal VTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTTY Uniprot no:CCNQDHCNKIELPTTVKSSPGLGPVELAAVIAGPVCFVCISLMLMVYICH P36897NRTVIHHRVPNEEDPSLDRPFISEGTTLKDLIYDMTTSGSGSGLPLLVQRT version numberIARTIVLQESIGKGRFGEVWRGKWRGEEVAVKIFSSREERSWFREAEIYQ 1 as of Jun. 1,TVMLRHENILGFIAADNKDNGTWTQLWLVSDYHEHGSLFDYLNRYTVT 1994VEGMIKLALSTASGLAHLHMEIVGTQGKPAIAHRDLKSKNILVKKNGTCCIADLGLAVRHDSATDTIDIAPNHRVGTKRYMAPEVLDDSINMKHFESFKRADIYAMGLVFWEIARRCSIGGIHEDYQLPYYDLVPSDPSVEEMRKVVCEQKLRPNIPNRWQSCEALRVMAKIMRECWYANGAARLTALRIKKTLS QLSQQEGIKM  8 HumanMNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDV VEGFAYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPT Uniprot no:EESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQEKKSVRG P15692KGKGQKRKRKKSRYKSWSVYVGARCCLMPWSLPGPHPCGPCSERRKH version numberLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR 2 as of Nov. 16, 2001  9 HUMANMGTSHPAFLVLGCLLTGLSLILCQLSLPSILPNENEKVVQLNSSFSLRCFG Platelet-ESEVSWQYPMSEEESSDVEIRNEENNSGLFVTVLEVSSASAAHTGLYTCY derived growthYNHTQTEENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIPCR factor receptorTTDPETPVTLHNSEGVVPASYDSRQGFNGTFTVGPYICEATVKGKKFQTI alphaPFNVYALKATSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDLQWTY Uniprot no:PGEVKGKGITMLEEIKVPSIKLVYTLTVPEATVKDSGDYECAARQATRE P16234,VKEMKKVTISVHEKGFIEIKPTFSQLEAVNLHEVKHFVVEVRAYPPPRIS version numberWLKNNLTLIENLTEITTDVEKIQEIRYRSKLKLIRAKEEDSGHYTIVAQNE 1 as of Apr. 1,DAVKSYTFELLTQVPSSILDLVDDHHGSTGGQTVRCTAEGTPLPDIEWMI 1990CKDIKKCNNETSWTILANNVSNIITEIHSRDRSTVEGRVTFAKVEETIAVRCLAKNLLGAENRELKLVAPTLRSELTVAAAVLVLLVIVIISLIVLVVIWKQKPRYEIRWRVIESISPDGHEYIYVDPMQLPYDSRWEFPRDGLVLGRVLGSGAFGKVVEGTAYGLSRSQPVMKVAVKMLKPTARSSEKQALMSELKIMTHLGPHLNIVNLLGACTKSGPIYIITEYCFYGDLVNYLHKNRDSFLSHHPEKPKKELDIFGLNPADESTRSYVILSFENNGDYMDMKQADTTQYVPMLERKEVSKYSDIQRSLYDRPASYKKKSMLDSEVKNLLSDDNSEGLTLLDLLSFTYQVARGMEFLASKNCVHRDLAARNVLLAQGKIVKICDFGLARDIMHDSNYVSKGSTFLPVKWMAPESIFDNLYTTLSDVWSYGILLWEIFSLGGTPYPGMMVDSTFYNKIKSGYRMAKPDHATSEVYEIMVKCWNSEPEKRPSFYHLSEIVENLLPGQYKKSYEKIHLDFLKSDHPAVARMRVDSDNAYIGVTYKNEEDKLKDWEGGLDEQRLSADSGYIIPLPDIDPVPEEEDLGKRNRHSSQTSEESAIETGSSSSTFIKREDETIEDIDMMDDIGIDSSDLVEDSFL 10 Human Ang-1MTVFLSFAFLAAILTHIGCSNQRRSPENSGRRYNRIQHGQCAYTFILPEHD Uniprot no:GNCRESTTDQYNTNALQRDAPHVEPDFSSQKLQHLEHVMENYTQWLQ Q15389KLENYIVENMKSEMAQIQQNAVQNHTATMLEIGTSLLSQTAEQTRKLTD versionVETQVLNQTSRLEIQLLENSLSTYKLEKQLLQQTNEILKIHEKNSLLEHKI number 2 as ofLEMEGKHKEELDTLKEEKENLQGLVTRQTYIIQELEKQLNRATTNNSVL Jan. 1,QKQQLELMDTVHNLVNLCTKEGVLLKGGKREEEKPFRDCADVYQAGF 1998NKSGIYTIYINNMPEPKKVFCNMDVNGGGWTVIQHREDGSLDFQRGWKEYKMGFGNPSGEYWLGNEFIFAITSQRQYMLRIELMDWEGNRAYSQYDRFHIGNEKQNYRLYLKGHTGTAGKQSSLILHGADFSTKDADNDNCMCKCALMLTGGWWFDACGPSNLNGMFYTAGQNHGKLNGIKWHYFKGPSYS LRSTTMMIRPLDF 11Human HGF MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTL Uniprot no:IKIDPALKIKTKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQCLWF P14210PFNSMSSGVKKEFGHEFDLYENKDYIRNCIIGKGRSYKGTVSITKSGIKCQ versionPWSSMIPHEHSFLPSSYRGKDLQENYCRNPRGEEGGPWCFTSNPEVRYE number 2 as ofVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTPHRHKFLPE Aug. 1, 1991RYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPLETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGSESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNMEDLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNLDHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRDLKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLPNYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAGAEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKIILTYKVPQS 12 PDGFB humanMNRCWALFLSLCCYLRLVSAEGDPIPEELYEMLSDHSIRSFDDLQRLLHG Uniprot no:DPGEEDGAELDLNMTRSHSGGELESLARGRRSLGSLTIAEPAMIAECKTR P01127TEVFEISRRLIDRTNANFLVWPPCVEVQRCSGCCNNRNVQCRPTQVQLR versionPVQVRKIEIVRKKPIFKKATVTLEDHLACKCETVAAARPVTRSPGGSQEQ number 1 as ofRAKTPQTRVTIRTVRVRRPPKGKHRKFKHTHDKTALKETLGA Jul. 21, 1986 13 Human IL-10MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFS Uniprot no:RVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQ P22301AENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAF versionNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN number 1 as of Aug. 1, 1991

Experimental Examples

1. Cryopreservation of Umbilical Cord Tissue Prior to Isolation ofMesenchymal Stem Cells

Umbilical cord tissue (the umbilical cords were donated with informedconsent of the mother) was processed for the subsequent isolation of themesenchymal stem cells from the amniotic membrane of the umbilical cordas follows.

1.1 Washing of Umbilical Cord Tissue Sample:

a. Remove scalpels from the protective cover.b. Hold the umbilical cord securely using the forceps and cut the cordinto a 10 cm length piece using a scalpel. Place the unused cord back inthe original tissue cup.c. Transfer the 10 cm long umbilical cord piece into a new 150 mmculture dish. The 150 mm culture dish may be used in place of the cups.d. Use the cover of the 150 mm culture dish as a resting place forforceps and scalpel.e. Remove 25 ml Plasmalyte A (Baxter, Catalog #2B2543Q) with a 30 mlsyringe. Hold the syringe at a 45° angle using one hand and dispense thePlasmalyte A directly onto the umbilical cord tissue.f. Holding the culture dish at a slight angle remove the Plasmalyte Awith a 30 ml syringe and blunt needle.g. Collect used Plasmalyte A in a 300 ml transfer bag that serves as atrash container and dispose it in the biohazard bin.h. Repeat wash procedure, if necessary using a new culture dish for eachwash. Make sure all blood clots on the surface have been removed. MorePlasmalyte A can be used if needed to clean the tissue.i. Place the tissue into a new labeled tissue culture dish to continuecutting the tissue. Place 20 ml of Plasmalyte A into the dish so thetissue does not dry out while cutting it.j. Cut the cords into equal approximately 1-cm sections resulting in 10sections in total.k. Further cut each 1 cm section into smaller pieces with approximately0.3 cm×0.3 cm to 0.5 cm×0.5 cm per section.l. Remove any Plasmalyte A that is in the dish.m. Pull 25 ml Plasmalyte A with a 30 ml syringe from the originalPlasmalyte A bag and dispense directly on the umbilical cord tissuepieces.n. Hold culture dish in an angle to collect all Plasmalyte A used forwashing the tissue on one side and remove it with a syringe and bluntneedle.o. Repeat wash one more time. There should not be any clots left.

NOTE: If the cord is not frozen right away, the umbilical cord tissue iskept in Plasmalyte A until ready to freeze.

1.2 Cryopreservation of Umbilical Cord Tissue:

a. Prepare cryopreservation solution:i. Prepare 50 ml freezing solution consisting of 60% Plasmalyte A, 30%of 5% Human Serum Albumin, and 10% dimethyl sulfoxide (DMSO).ii. Label a 150 ml transfer bag with “Tissue freeze solution” and attacha plasma transfer set to the port using aseptic technique.iii. Remove 30 ml Plasmalyte A with a 30 ml Syringe from the originalPlasmalyte A bag and transfer it in the transfer bag labeled “tissuefreeze solution” with the time and date solution is made.iv. Remove 15 ml of 5% Human Serum Albumin with a 20 ml syringe andtransfer it into the labeled transfer bag.v. Add 5 ml DMSO to the transfer bag.vi. Mix well and record mixing of freeze solutionb. Remove the Plasmalyte A from the tissue before adding the freezesolution.c. Using a 60 ml syringe, pull all 50 mls of the freeze solution intothe syringe add approximately 30 ml freeze solution to the 150 mm cellculture dish containing the umbilical cord tissue. Place a blunt needleon the syringe to keep it sterile.d. Swirl the culture dish containing the tissue and freezing solutionevery minute for 10 minutes.e. Using forceps, select 8 randomly chosen sections and place them ineach of the four 4 ml cryovials. Select 4 randomly chosen sections andplace them into one 1.8 ml cryovial. These sections should be free ofblood clots.f. Fill each cryovial containing the umbilical cord tissue with theremaining freezing solution to the 3.6 ml filling line for the 4 mltubes and the 1.8 ml line for the 1.8 ml Nunc vial.g. Label one Bactec Lytic/10—Anaerobic/F and one Bactec Plus Aerobic/Fbottle with tissue ID.h. Remove 20 ml freeze solution from the culture dish with a syringe anda blunt needle, after wiping the Bactec vials with an alcohol swab,switch the blunt needle for an 18 g needle and inoculate the aerobic andthe anaerobic Bactec bottles with 10 ml each.i. Start controlled rate freezer.j. After controlled rate freeze is completed place the units in acontinuous temperature monitored liquid nitrogen freezer until furtheruse.

2. Isolation of Mesenchymal Cord Lining Stem Cells from Umbilical CordTissue

2.1. Preparing Media for Processing MSCs from Umbilical Cord Tissue:

a. To make 500 ml PTT6 (culture/growth media) add the following in theorder listed:i. DMEM, 250 mlii. M171 118 mliii. DMEM F12 118 mliv. FBS 12.5 ml (final concentration of 2.5%)v. EGF 1 ml (final concentration of 10 ng/ml)vi. Insulin 0.175 ml (final concentration of 5 μg/ml)

The above-mentioned volumes of components i. to vi when result in afinal volume of 499.675 ml culture medium. If no further components areadded to the culture medium, the remaining 0.325 ml (to add up to avolume of 500 ml) can, for example, be any of components i. to iv, thatmeans either DMEM, M171, DMEM/F12 or FBS. Alternatively, theconcentration of the stock solution of EGF or Insulin can of course beadjusted such that the total volume of the culture medium is 500 ml.Alternatively, a stock solution of an antibiotic such asPenicillin-Streptomycin-Amphotericin can be added to result in a finalvolume of 500 ml. It is also possible to add to the culture medium avolume of 0.325 ml of one or more of the following supplements: adenine,hydrocortisone, 3,3′,5-Triiodo-L-thyronine sodium salt (T3), therebyreaching a total volume of 500 ml culture medium.

vii. Label the bottle “PTT6” with date media was prepared, initial ofthe operator, and the phrase “expires on” followed by the expirationdate. Expiration date is the earliest expiration date of any of thecomponent or 1 month from the preparation date, whichever comes first.b. To make the rinse media (Hank's Buffered Salt Solution (HBSS) withoutCalcium or Magnesium and with 5% FBS), add 2.5 ml FBS to 47.5 ml of HBSSin a 50 ml centrifuge tube. Label the tube “Rinse Media” with operatorinitials and date the media is made.c. All media will be tested for sterility using BactecLytic/10—Anaerobic/F (Becton Dickinson & Company) and BactecPluc+Aerobic/F (Becton Dickinson & Company). Inject 20 ml of preparedmedia into each bottle.

2.2 Thawing of Umbilical Cord Tissue for MSC Harvesting:

a. Initiate the thaw once an operator is prepared to process the samplein the clean room. Do not thaw more than 1 vial at a time unless thevials originate from the same donor.b. Wipe the water bath with disinfectant followed by 70% isopropanol andfill it with 1 L sterile water. Heat the water bath up to 36-38° C.c. Prepare 10 mL of rinse medium consisting of 70% to 90% PlasmaLyte Ain the clean room under a biosafety cabinet. Sterile filter the solutionwith a 0.2-μm syringe filter attached to a 10 ml syringe and keep thesolution refrigerated until use.d. Place a processing label on a 50 ml conical tube.e. Confirm water bath temperature is at 36-38° C.f. Take vial(s) of tissue from the liquid nitrogen storage and thawrapidly in the 37° C. water bath filled with 1 L of sterile water. Thevial holder for the Mr. Frosty Nalgene Cryo 1° C. freezing containerfloats with vials in place and can be used as a floating rack whenthawing samples.g. Remove the vial from the water bath and spray them with 70%Isopropanol solution. A good time to pull the vial from the water bathis when small ice can be seen floating in the vial—suggest internaltemperature of the vial is less than 37° C.h. Place vial into pass-through and alert the clean room processingtechnician.

2.3 Preparing for Tissue Processing:

a. Umbilical cord tissue processing should be performed in anenvironmentally monitored (EM) clean room. At the end of each shift,full room and hood cleaning are performedb. Prepare/clean the biosafety cabinet.c. Perform viable particle counting while working in the biosafetycabinet.d. Assemble all necessary supplies in the biosafety cabinet checkingeach for packaging damage and expiration dates. When handling syringes,serological pipets, sterile forceps, scalpels, tissue plates, andneedles, make sure not to touch any surface that will come in contactwith the sterile product. Only the exterior of the syringe barrel,tubing, plunger tip and/or needle cap or sheath may be safely handled.Discard supply if the surface has been touched or has touched anon-sterile surface.e. Record lot numbers and expiration dates (if applicable) of allreagents and supplies to be used.f. Receive the thawed vial by cleaning the vial with lint-free wipemoistened with 70% alcohol before transferring into the biosafetycabinet.g. Using an aspirating needle with a syringe, withdraw as much liquidfrom the vial. Avoid suctioning the tissue.h. Using sterile forceps, transfer the tissue into a sterile 100 mmpetri dish.i. Add an aliquot of 5 ml rinse medium to the tissue fragments.j. Swirl the contents for 15-30 seconds, then remove the rinse mediumwith a pipette or syringe with aspirating needle. Repeat this rinseprocess twice.k. Add 2 mL of rinse medium to the tissue to avoid drying out thetissue.

2.4. Initiating MSC Outgrowth from Tissue:

a. Label the bottom of a 6-well plate “Outgrowth 1” with MSC lot numberor umbilical cord tissue ID and the date outgrowth is initiated. If 60mm tissue culture dish is used, divide the plate into 4 quadrants bydrawing a grid on the bottom of the dish.b. Using sterile, disposable forceps, place one 3×3 mm to 5×5 mm tissueinto each well. If using a 60 mm tissue culture dish, place the tissueinto the middle of each quadrant to keep the tissues apart (more than 1cm from each other).c. Fill each well with 3 ml of PTT6.d. Using an aspirating needle coupled to 30 ml syringe, withdraw enoughmedia to barely cover the tissue. Do not tilt the plate. Do not touchthe bottom of the well with the aspirating needle.e. Using an inverted light microscope, observe for cell outgrowth everyday (24±6 hrs). Real time cell culture imaging system may be used inplace of the light microscope.f. Change media every day. Be sure to equilibrate the media to roomtemperature before use.i. Aspirate off the medium.ii. Add 3 ml of PTT6.iii. Aspirate until tissue is barely submerged in the medium.g. When cellular outgrowth is observed from the tissue, transplant thetissue to a new 6-well plate using the same procedure as 4.a to 4.eabove except label the plate “Outgrowth 2”. Maintain cell outgrowth in“Outgrowth 1” plate by adding 2 ml of PTT6 to each well. Observe forconfluency every day. Replace media every 2-3 days (be sure toequilibrate the media to room temperature before use).h. When cell outgrowth is observed in “Outgrowth 2” plate, repeat step4.a to 4.e except label the plate “Outgrowth 3.” Maintain cell outgrowthin “Outgrowth 2” plate by adding 2 ml of PTT6 to each well. Observe forconfluency every day. Replace media every 2-3 days (be sure toequilibrate the media to room temperature before use).i. When outgrowth is observed in “Outgrowth 3” plate, discard thetissue. If the tissues are very small and do not seem to interfere withcell growth, dispose of the tissue when subculturing.j. When cells reach 40-50% confluency, observe cells every days toprevent over-expansion.k. When cells reach 70-80% confluency, subculture the cells. Do notallow cells to expand beyond 80% confluence.

With the size of the tissue explants being about 1-3 mm, and the tissueexplant/cell culture is performed in 175 mm squared culture dishes, theaverage number of mesenchymal stem cells harvested from an explant istypically about 4,000-6,000 cells/explant. Accordingly, when themesenchymal stem cells are simultaneously grown out of 48 explants about300,000 cells can be obtained at harvest. These 300,000 mesenchymal stemcells collected from explants can then be used for subculturing byseeding a 175 cm² cell culture flask with such 300,000 cells asdescribed in the following Example 2.5 (this can be referred to asPassage 1). The mesenchymal stem cells obtained from this passage 1 canthen be used to seed again 175 cm² flasks (Passage 2) and expand thecells as described in the following Example 2.5. The cells obtained fromboth Passage 1 and Passage 2 can be “banked” by cryo-preservation, withthe mesenchymal stem cells obtained after Passage 2 being considered torepresent the Master Cell Bank which will be for further expansion ofthe mesenchymal stem cells, for example, in a bioreactor as explainedbelow in Example 2.7.

2.5. Subculturing MSC in Cell Culture Dishes

a. Perform viable particle while working in the biosafety cabinet.Equilibrate all media to room temperature before use.b. When cell outgrowth reaches about 70-80% confluency, subculturecells.i. Remove PTT6 from the petri dish.ii. Rinse with HBSS without Calcium or Magnesium.iii. Add 0.2 ml 1× TrypLE-EDTA and swirl for 1-2 minutes.iv. Tilt the dish 30-45° to allow cells to shift down by gravitationalflow. Gentle tapping on the side of the plate expedites detachment.v. Add 1 ml of PTT6. Pipette up and down gently then transfer cells to a15 ml centrifuge tube. Use clean pipette tip with each well. Cells fromall 6 wells can be pooled into a single 15 ml tube.vi. Centrifuge for 10 minutes at 1200 rpm.vii. Remove supernatant and resuspend cells with 5 ml PTT6.c. Subculturing MSCi. Aliquot 50 μl of the cell suspension and assay for TNC and viabilityby Trypan Blue Exclusion Assay.ii. Count cells using a hemocytometer. Expect to count 20-100cells/square. If the count higher than 100, dilute the original sample1:5 and repeat Trypan Blue method using a hemocytometer.iii. Calculate viable cells/ml and total viable cells:1. Viable cells/ml=viable cell count×dilution factor×10⁴2. Total viable cells=viable cell count×dilution factor×total volume×10⁴iv. Calculate % viability:1. % viability=viable cell count×100/(viable cell count+dead cell count)v. Dilute the cell suspension to 1.0×10⁶ cells/ml:

-   -   1. “X” volume=Total viable cells/10⁶ cells/ml    -   2. For example, if total viable cell number is 1.0×10⁷;    -   3. “X”=10⁷/10⁶ cells/ml or 10 ml, therefore, you would bring        your total cell volume up to 10 ml by adding 5 ml to your cell        suspension (that is at 5 ml).        vi. If the cell suspension is less than 106/ml, determine the        volume required to seed 2×106 cells for each 150 mm petri dish        or 175 cm2 flask.        1. Volume for 2×10⁶ cells=2×10⁶ cells÷viable cells/ml        2. For example, if viable cells/ml is 8×10⁵ cells/ml, 2×10⁶        cells÷ 8×10⁵ cells/ml or 2.5 ml are needed.        vii. Set aside 0.5 ml for MSC marker analysis.        viii. Seed 2×10⁶ cells to each 150 mm petri dish or 175 cm²        flask with 30 ml PTT6.        ix. Observe cells for attachment, colony formation, and        confluence every three days. When cells reach 40-50% confluence,        observe cells every one-two days to prevent over-expansion. DO        NOT allow cells to expand beyond 80% confluence. A real time        cell culturing monitoring system can be used in place of the        light microscope.        x. Replace media every 2-3 days.

2.6 Cryopreserving MSC Cells

a. Perform viable particle while working in the biosafety cabinet.b. When cells reach 70-80% confluence, detach cells using 2 ml 1×TrypLE-EDTA for each 150 mm petri dish or 175 cm2 flask.i. Remove PTT6 from the petri dish.ii. Wash with 5 ml HBSS or PBS without calcium or magnesium.iii. Add 2 ml 1× TrypLE-EDTA and swirl for 1-2 minutes.iv. Tilt the dish 30-45° to allow cells to shift down by gravitationalflow. Gentle tapping on the side of the petri dish helps to expeditedetachment.v. Add 10 ml PTT6 to inactivate TrypLE. Mix well to dissociate cellclumps.vi. Transfer cells to 15 ml centrifuge tube using a Pasteur pipette.vii. Centrifuge for 10 minutes at 1200 rpm.viii. Aspirate medium and resuspend with 10 ml PTT6.ix. Aliquot 50 μl and determine total viable cell number and % viabilityas above. Cell count will need to be performed within 15 minutes as thecells may start clumping.c. Preparing cells for cryopreservationi. Prepare Cell Suspension Media and Cryopreservation Media and freezethe cells

2.7. Subculturing (Expansion) of MSC in a Quantum Bioreactor (TerumoBTC, Inc.)

It is also possible to use a Quantum Bioreactor can used to expand theMSC. The starting cell number for the expansion in the QuantumBioreactor should range between 20 to 30 million cells per run. Thetypical yield per run is 300 to 700 million MSC at harvest. TheBioreactor is operated following the protocol of the manufacturer. Theso obtained mesenchymal stem cells are typically cryo-preserved (seebelow) and serve as Working Cell Bank.

Materials/Reagents:

1. Quantum Expansion Set 2. Quantum Waste Bag 3. Quantum Media Bag 4.Quantum Inlet Bag 5. PTT6 6. PBS 7. Fibronectin 8. TrypLE

9. 3 ml syringe10. Glucose test strips11. Lactate test strips12. 60 ml Cell Culture Plate or equivalent

13. Medical Grade 5% CO₂ Gas-mix 14. 50 ml Combi-tip

Equipment:

1. Biosafety Cabinet 2. Glucose Meter (Bayer Healthcare/Ascensia ContourBlood Glucose Meter) 3. Lactate Plus (Nova Biomedical)

4. Peristaltic pump with head

5. Centrifuge, Eppendorf 5810 6. Sterile Tube Connector 7. M4 RepeatPipettor 8. RF Sealer

Procedure:

1. Preparing the Quantum Bioreactor

-   -   a) Priming the Quantum Bioreactor    -   b) Coating the bioreactor:        -   1) Prepare the fibronectin solution in the biosafety            cabinet.            -   1) Allow lyophilized fibronectin to acclimate to room                temperature (≥15 min at room temperature)            -   2) Add 5 ml of sterile distilled water; do not swirl or                agitate            -   3) Allow fibronectin to go into solution for 30 min.            -   4) Using a 10 ml syringe attached with an 18 g needle,                transfer the fibronectin solution to a cell inlet bag                containing 95 ml of PBS.        -   2) Attach the bag to the “reagent” line        -   3) Check for bubbles (bubbles may be removed by using            “Remove IC Air” or “Remove EC Air” and using “Wash” as the            inlet source.        -   4) Open or set up program for coating the bioreactor (FIG. 1            . Steps 3-5).        -   5) Run the program        -   6) While the program is running to coat the bioreactor,            prepare a media bag with 4 L of PTT6 media.        -   7) Attach the media bag to the IC Media line using a sterile            tube connector.        -   8) When the bioreactor coating steps are completed, detach            the cell inlet bag used for fibronectin solution using a RF            sealer.    -   c) Washing off excess fibronectin    -   d) Conditioning the bioreactor with media

2. Culturing the Cells in the Quantum Bioreactor

-   -   a) Loading and attaching the cells with Uniform Suspension:    -   b) Feeding and cultivation of the cells        -   1) Chose media flow rate to feed the cells.        -   2) Sample for lactate and glucose every day.        -   3) Adjust the flow rate of the media as the lactate levels            increase. The actual maximal tolerable lactate concentration            will be defined by a flask culture from which the cells            originate. Determine if adequate PTT6 media is in the media            bag. If necessary, replace the PTT6 media bag with a fresh            PTT6 media bag.        -   4) When the flow rate has reached the desired value, measure            lactate level every 8-12 hours. If the lactate level does            not decrease or if the lactate level continues to increase,            harvest the cells.            3. Harvesting the Cells from the Quantum Bioreactor    -   a) When lactate concentration does not decrease, harvest the        cells after sampling for lactate and glucose for the last time.    -   b) Harvesting the cells:        -   1) Attach cell inlet bag filled with 100 ml TrypLE to the            “Reagent” line using a sterile tube connector.        -   2) Confirm sufficient PBS is in the PBS bag. If not, attach            a new bag with at least 1.7 liters of PBS to the “Wash” line            using a sterile tube connector.        -   3) Run the Harvest program

4. Cryopreserving the Cells

-   -   1) Once the cells have been harvested, transfer the cells to 50        ml centrifuge tube to pellet the cells.    -   2) Resuspend using 25 ml of cold cell suspension solution. Count        the cells using Sysmex or Biorad Cell counter. Attach the cell        count report to the respective Quantum Processing Batch Record.    -   3) Adjust cell concentration to 2×10⁷/ml    -   4) Add equal volume cryopreservation solution and mix well (do        not shake or vortex)    -   5) Using a repeat pipettor, add 1 ml of the cell suspension in        cryopreservative to each 1.8 ml vial. Cryopreserve using the CRF        program as described in the SOP D6.100 CB Cryopreservation Using        Controlled Rate Freezers    -   6) Store the vials in a designated liquid nitrogen storage        space.    -   7) Attach the CRF run report to the form respective MSC        P3-Quantum Processing Batch Record.

3. Analysis of Stem Cell Marker Expression in Mesenchymal Cord LiningStem Populations Isolated from Umbilical Cord Tissue, Using DifferentCulture Media

Flow cytometry experiments were carried out to analyse mesenchymal stemcells isolated from the umbilical cord for the expression of themesenchymal stem cell markers CD73, CD90 and CD105.

For these experiments, mesenchymal stem cells were isolated fromumbilical cord tissue by cultivation of the umbilical cord tissue inthree different cultivation media, followed by subculturing of themesenchymal stem cells in the respective medium as set forth in Example2.

The three following culture media were used in these experiments: a) 90%(v/v/DMEM supplemented with 10% FBS (v/v), b) the culture medium PTT-4described in US patent application US 2008/0248005 and the correspondingInternational patent application WO2007/046775 that consist of 90% (v/v)CMRL1066, and 10% (v/v) FBS (see paragraph [0183] of WO2007/046775) andc) the culture medium of the present invention PTT-6 the composition ofwhich is described herein. In this flow cytometry analysis, twodifferent samples of the cord lining mesenchymal stem cell (CLMC)population were analysed for each of the three used culture media.

The following protocol was used for the flow cytometry analysis.

Materials and Methods

Instruments name Company Name Serial Name BD FACS CANDO BD V07300367Inverted Microscope, CKX41SF Olympus 4K40846 Centrifuge, Micro spinTabletop Biosan 010213-1201-0003

Reagent list Company Name CatLog Number 10 × Trypsin Biowest X0930-10010 × PBS Lonza 17-517Q DMEM Lonza 12-604F Fetal Bovine Serum GEhealthcare A11-151

Antibody list Company Name CatLog Number Human CD73 Purified AD2 0.1 BD550256 mg Human CD90 Purified 5E10 1 mL BD 550402 Human CD105 Purified266 0.1 BD 555690 mg Alexa Fluor 647 goat anti-mouse BD A21235 IgG (H +L) *2 mg/mL*

Reagents name Composition 1 × PBS (1 L) 100 ml of 10 × PBS + 900 ml ofsterile distilled H20 1 × PBA (50 ml) 49.5 ml of 1 × PBS + 0.5 ml of FBS

Procedure

a) Cell Isolation and Cultivation from the Umbilical Cord LiningMembrane

-   -   1. Explant tissue samples were incubated in a cell culture plate        and submerged in the respective medium, then keep it in CO₂        incubator at 37° C. as explained in Example 2.    -   2. The medium was changed every 3 days.    -   3. Cell outgrowth from tissue culture explants was monitored        under light microscopy.    -   4. At a confluence of about 70%, cells were separated from dish        by trypsinization (0.0125% trypsin/0.05% EDTA) and used for flow        cytometry experiments.

b) Trypsinization of Cells for Experiments

-   -   1. Remove medium from cell culture plate    -   2. Gently rinse with sterile 1×PBS to remove traces of FBS as        FBS will interfere with the enzymatic action of trypsin.    -   3. Add 1× trypsin to cell culture plate and incubate for 3-5 min        in 37° C.    -   4. Observe cells under microscope to ensure that they are        dislodged. Neutralize trypsin by adding complete media        containing FBS (DMEM with 10% FBS).    -   5. Use a pipette to break up cell clumps by pipetting cells in        media against a wall of the plate. Collect and transfer cell        suspension into 50 ml centrifuge tubes    -   6. Add sterile 1×PBS to plate and rinse it, Collect cell        suspension into the same centrifuge tube.    -   7. Centrifuge it at 1800 rpm for 10 mins.    -   8. Discard supernatant and re-suspend cell pellet with PBA        medium.

c) Counting Cells

-   -   1. Ensure that the haemocytometer and its cover slip are clean        and dry, preferably by washing them with 70% ethanol and letting        them dry before wiping them with Kim wipes (lint-free paper).    -   2. Aliquot a small amount of cells in suspension into a micro        centrifuge tube and remove from the BSC hood.    -   3. Stain cells in suspension with an equal volume of Trypan        Blue, e.g. to 500 μl of suspension add 500 μl of Trypan Blue        (dilution factor=2×, resulting in 0.2% Trypan Blue solution).    -   4. Avoid exposure of cells to Trypan Blue for longer than 30        mins as Trypan Blue is toxic and will lead to an increase in        non-viable cells, giving a false cell count.    -   5. Add 20 μl of the cell suspension mixture to each chamber of a        haemocytometer and view under a light microscope.        -   a. Count the number of viable cells (bright cells;            non-viable cells take up Trypan Blue readily and thus are            dark) in each quadrant of the haemocytometer for a total of            8 quadrants in the upper and lower chamber.            -   Total cell count is given as (Average number of                cells/quadrant)×10⁴ cells/ml.

d) Staining Cells

i. Preparation Before Staining Cells

-   -   Cell suspension are aliquot into 3 tubes (CD73, CD90, CD105) in        duplicates and 2 tubes (negative control), each containing        50,000 cells.

ii. Staining with Primary Antibody (Ab)

-   -   Add 1 μl [0.5 mg/ml Ab] of primary antibody to 100 ul cell        suspension. Incubate at 4° C. for 45 min.    -   Make up to 1 ml with PBA.    -   Centrifuge 8000 rpm at 4° C. for 5 mins.    -   Remove supernatant.    -   Add 1 ml of PBA and re-suspend pellet    -   Centrifuge 8000 rpm at 4° C. for 5 mins.    -   Remove supernatant.    -   Re-suspend in 100 ul PBA.

iii. Staining with Secondary Ab—in the Dark

-   -   Add 1 ul [0.5 mg/ml ab] of secondary antibody to 100 ul cell        suspension. Incubate at 4° C. for 30 min.    -   Make up to 1 ml with PBA.    -   Centrifuge 8000 rpm at 4° C. for 5 mins.    -   Remove supernatant.    -   Add 1 ml of PBA and re-suspend pellet    -   Centrifuge 8000 rpm at 4° C. for 5 mins.    -   Remove supernatant    -   Re-suspend in 200-300 ul PBA for flow cytometry analysis    -   Transfer cells to FACS tube for reading in BD FACS CANDO flow        cytometry.

The results of the flow cytometry analysis are shown in FIG. 6 a to FIG.6 c . FIG. 6 a shows the percentage of isolated mesenchymal cord liningstem cells expressing stem cell markers CD73, CD90 and CD105 afterisolation from umbilical cord tissue and cultivation in DMEM/10% FBS,FIG. 6 b shows the percentage of isolated mesenchymal cord lining stemcells expressing stem cell markers CD73, CD90 and CD105 after isolationfrom umbilical cord tissue and cultivation in PTT-4 and FIG. 6 c showsthe percentage of isolated mesenchymal cord lining stem cells expressingstem cell markers CD73, CD90 and CD105 after isolation from umbilicalcord tissue and cultivation in PTT-6. As can be seen from FIG. 6 a , thepopulation isolated using DMEM/10% FBS as culture medium cultivation hasabout 75% CD73+ cells, 78% CD90+ cells and 80% CD105+ cells (average oftwo experiments), while after isolation/cultivation of umbilical cordtissue using PTT-4 culture medium (see FIG. 6 b ) the number ofmesenchymal stem cells that are CD73-positive, CD90-positive andCD105-positive are about 87% (CD73+ cells), 93%/CD90+ cells) and 86%(CD105+ cells) average of two experiments. The purity of the mesenchymalstem cell population that was obtained by means of cultivation in thePTT-6 medium of the present invention is at least 99.0% with respect toall three markers (CD73, CD90, CD105), meaning the purity of this cellpopulation is significant higher than for cultivation using PTT-4 mediumor DMEM/10% FBS. In addition and even more importantly, the mesenchymalstem cell population obtained by means of cultivation in PTT-6 isessentially a 100% pure and defined stem cell population. This makes thestem cell population of the present invention the ideal candidate forstem cell based therapies. Thus, this population of mesenchymal cordlining stem cells may become the gold standard for such stem cell basedtherapeutic approaches.

The findings shown in FIG. 6 are further corroborated by the results ofthe flow cytometry analysis that are shown in FIG. 7 a and FIG. 7 b .FIG. 7 a shows the percentage of isolated mesenchymal cord lining stemcells (mesenchymal stem cells of the amniotic membrane of umbilicalcord) that express the stem cell markers CD73, CD90 and CD105 and lackexpression of CD34, CD45 and HLA-DR after isolation from umbilical cordtissue and cultivation in PTT-6 medium. As shown in FIG. 7 a , themesenchymal stem cell population contained 97.5% viable cells of which100% expressed each of CD73, CD90 and CD105 (see the rows “CD73+CD90+”and “CD73+CD105+”) while 99.2% of the stem cell population did notexpress CD45 and 100% of the stem cell population did not express CD34and HLA-DR (see the rows “CD34+CD45− and “CD34−HLA-DR−). Thus, themesenchymal stem cells population obtained by cultivation in PTT-6medium is essentially a 100% pure and defined stem cell population thatmeets the criteria that mesenchymal stem cells are to fulfill to be usedfor cell therapy (95% or more of the stem cell population express CD73,CD90 and CD105, while 98% or more of the stem cell population lackexpression of CD34, CD45 and HLA-DR, see Sensebe et al. “Production ofmesenchymal stromal/stem cells according to good manufacturingpractices: a review”, supra). It is noted here that the presentmesenchymal stem cells of the amniotic membrane adhere to plastic instandard culture conditions and differentiate in vitro into osteoblasts,adipocytes and chondroblasts, see U.S. Pat. Nos. 9,085,755, 8,287,854 orWO2007/046775 and thus meet the criteria generally accepted for use ofmesenchymal stem cells in cellular therapy.

FIG. 7 b shows the percentage of isolated bone marrow mesenchymal stemcells that express CD73, CD90 and CD105 and lack expression of CD34,CD45 and HLA-DR. As shown in FIG. 7 b , the bone marrow mesenchymal stemcell population contained 94.3% viable cells of which 100% expressedeach of CD73, CD90 and CD105 (see the rows “CD73+CD90+” and“CD73+CD105+”) while only 62.8% of the bone marrow stem cell populationlacked expression of CD45 and 99.9% of the stem cell population lackedexpression CD34 and HLA-DR (see the rows “CD34−CD45− and “CD34−HLA-DR−).Thus, the bone marrow mesenchymal stem cells that are considered to begold standard of mesenchymal stem cells are by far less homogenous/purein terms of stem cell marker than the mesenchymal stem cells population(of the amniotic membrane of the umbilical cord) of the presentapplication. This finding also shows that the stem cell population ofthe present invention may be the ideal candidate for stem cell basedtherapies and may become the gold standard for stem cell basedtherapeutic approaches.

Experiments Showing that the Mesenchymal Stem Cell Population of theInvention can be Transported/Stored in HypoThermosol™:

To analyze health and viability of the mesenchymal stem cells asdescribed herein in different storage or transport carrier, twodifferent carriers were compared to each other. Namely, the carrierHypoThermosol™-FRS was compared to the carrier PlasmaLyte-A. Both arecommercially available. HypoThermosol™-FRS the product sheet of which isshown in FIG. 30 and its composition is described elsewhere herein. Each100 mL PlasmaLyte contains 526 mg of Sodium Chloride, USP (NaCl); 502 mgof Sodium Gluconate (C₆H₁₁NaO₇); 368 mg of Sodium Acetate Trihydrate,USP (C₂H₃NaO₂.3H₂O); 37 mg of Potassium Chloride, USP (KCl); and 30 mgof Magnesium Chloride, USP (MgCl₂.6H₂O). PlasmaLyte does not containantimicrobial agents. The pH of PlasmaLyte is adjusted with sodiumhydroxide to 7.4 (6.5 to 8.0).

The experimental setup for comparison is shown in FIG. 8 . First themesenchymal stem cell population as described herein were outgrown incell culture flasks. The number of living mesenchymal stem cells wascounted and then 2 million cells/vial were stored for different periodsof time in either PlasmaLyte-A or Hypothermosol™-FRS. After storagecells have been counted in sample of ≤50 μl daily for days 1-5 (totalliquid withdrawal 250 μl) and checked for viability by staining thecells with Trypan blue. Further, on days 1, 3 and 5 sample ≤80 μl weretaken and analyzed. In addition, the supernatant was obtained andfrozen. Then PDGF-AA, PDGF-BB, VEGF, IL-10, Ang-1, HGF and TGFβ1 weremeasured by FLEXMAP 3D system.

FIG. 9 summarizes viability data. As can be seen from the left-handgraph, 73% of the total cells (about 95%) with which the storing startedwere still viable 7 days after storage in HypoThermosol™. On thecontrary after 7 days of storage in PlasmaLyte-A only 42% of the totalof cells (about 94%) with which the storage started were still viable.All counts based on duplicate readings that are within 10% of oneanother (following SOP CR D2.600.1). During counting, cells stored inHypoThermosol™ were noticeably smaller with smooth and defined edges. Bycontrast, cells in Plasmalyte-A appeared of a range of sizes.HypoThermosol™ noticeably supports membrane integrity and presumablysurvival over a week timespan (6 days). Similar results are also shownin the graph of the right-hand side.

FIG. 10 shows the results obtained when measuring the cell diameter ofcells. The mesenchymal stem cell population as described herein whenkept in HypoThermosol™ are narrower in diameter range when compared tocells kept in PlasmaLyteA. Comparison took place after 3 days ofstorage.

FIG. 11 shows the TGFß1 concentration in supernatant from themesenchymal stem cell population as described herein stored inHypoThermosol™ or PlasmaLyte-A after 48 hrs of storage. As can be seenfrom the graph on the right-hand side, cells secrete about as much TGFß1when stored in HypoThermosol™ and when stored in PlasmaLyte-A. Ingeneral, over time, the amount of secreted TGFß1 decreased (graph on theright hand side).

FIGS. 12 and 13 show control experiments. Here, the PDGF-BB and IL-10concentration was measured in supernatent from mesenchymal stem cellpopulation as described herein stored in HypoThermosol™ or PlasmaLyte-Afor 48 hrs. Since PDGF-BB or IL-10 are not normally secreted by themesenchymal stem cell population as described herein, no PDGF-BB orIL-10 were detectable in any sample.

FIG. 14 shows the VEGF concentration in supernatant from mesenchymalstem cell population as described herein stored in HypoThermosol™ orPlasmaLyte-A for 48 hrs. As can be seen from the graph on the right-handside, cells secrete about as much VEGF when stored in HypoThermosol™ orPlasmaLyte-A on day 0. On day 1 and 5 cells secreted more VEGF whenstored in PlasmaLyte-A. Notably, when stored for 3 days cells secretedmore VEGF when stored in HypoThermosol™ than when stored inPlasmaLyte-A. Thus, HypoThermosol™ outperforms PlasmaLyte-A after day 3of storage. The more VEGF is detected the healther is the culture. Thus,by secreting more VEGF after 3 days storage in HypoThermosol™ than whenstored in PlasmaLyte-A, cells are healthier in HypoThermosol™ than inPlasmaLyte-A. From 5 days of storage onwards PlasmaLyte seems to becomea more favourable carrier, because at the time point 5 days, cellsstored in PlasmaLyte-A secreted more VEGF. In general, over time, theamount of secreted VEGF decreased (graph on the right hand side).

FIG. 15 shows the PDGF-AA concentration in supernatant from mesenchymalstem cell population as described herein stored in HypoThermosol™ orPlasmaLyte-A for 48 hrs. As can be seen from the graph on the right-handside, cells secrete about as much PDGF-AA when stored in HypoThermosol™than when stored in PlasmaLyte-A on day 0. On day 1 and 5 cells secretedmore PDGF-AA when stored in PlasmaLyte-A. Notably, when stored for 3days cells, secreted more PDGF-AA when stored in HypoThermosol™ thanwhen stored in PlasmaLyte-A. Thus, cells stored in HypoThermosol™ arehealthier than cells stored in PlasmaLyte-A after 3 days of storage.From 5 days of storage onwards, PlasmaLyte seems to become a morefavourable carrier, because at the time point 5 days cells stored inPlasmaLyte-A secreted more PDGF-AA. In general over time the amount ofsecreted PDGF-AA decreased (graph on the right hand side).

FIG. 16 shows the Ang-1 concentration in supernatant from mesenchymalstem cell population as described herein stored in HypoThermosol™ orPlasmaLyte-A for 48 hrs. As can be seen from the graph on the right-handside, cells secrete about as much Ang-1 when stored in HypoThermosol™ orPlasmaLyte-A on day 0 and 3. On day 5 cells secreted more Ang-1 whenstored in PlasmaLyte-A. Notably, when stored for 1 day, cells secretedmuch more Ang-1 when stored in HypoThermosol™ than when stored inPlasmaLyte-A. Thus, cells stored in HypoThermosol™ seem to be healthierthan when stored in PlasmaLyte-A for at least 48 hrs until 3 days ofstorage. From 5 days of storage onwards PlasmaLyte seems to become amore favourable carrier, because at this time point cells stored inPlasmaLyte-A secreted more Ang-1. In general, over time, the amount ofsecreted Ang-1 decreased (graph on the right hand side).

FIG. 17 shows the HGF concentration in supernatant from mesenchymal stemcell population as described herein stored in HypoThermosol™ orPlasmaLyte-A after 48 hrs of storage. As can be seen from the graph onthe right-hand side, cells secrete about as much HGF when stored inHypoThermosol™ than when stored in PlasmaLyte-A on day 0. On day 3 and 5cells secreted more HGF when stored in PlasmaLyte-A. Notably, whenstored for 1 day, cells secreted much more HGF when stored inHypoThermosol™ than when stored in PlasmaLyte-A. Thus, cells stored inHypoThermosol™ seem to be healthier than cells stored in PlasmaLyte-Abetween at least 1 day (48 hrs) until 3 days of storage. From 3 days onPlasmaLyte-A seems to become a more favourable carrier, because at thetime points 3 and 5 days cells stored in PlasmaLyte-A secreted more HGF.In general, over time, the amount of secreted HGF decreased (graph onthe right hand side).

In summary from the above data it can be concluded that storage of themesenchymal stem cell population of the present invention inHypoThermosol™ outperforms storage in PlasmaLyte-A especially for thefirst 3 days of storage.

Experiments Showing that the Mesenchymal Stem Cell Population of theInvention have Wound Healing Properties by Topical Treatment of Pigs:

Preclinical studies have also been performed using 10-week old femaleYorkshire-Landrace pigs (50 kg). The treatments were performed atSingHealth Experimental Medicine Centre in Singapore. The pigs wererendered diabetic with 120 mg/kg streptozotocin and allowed to recoverfor 45 days prior to creating six 5 cm×5 cm full thickness wounds ontheir backs (see FIG. 18 ). Pigs (n=2) were treated twice weekly with10⁵ human mesenchymal stem cell population as described herein per cm²for 4 weeks. The two control pigs were treated with PBS. Wounds werephotographed on postoperative day 0 (PODay 0) and every seven days untilpostoperative Day 35. The wounds were analyzed for surface area size byImageJ. By Day 35, the addition of mesenchymal stem cell population asdescribed herein had resulted in closure of 10 of 12 diabetic wounds(83%), compared to only 3 of 12 (25%) of the PBS-treated control wounds.The rate of wound healing was 0.8 cm²/day with the mesenchymal stem cellpopulation as described herein compared to 0.6 cm²/day in the controlanimals, an improvement of 33%. Results of this study are summarized inFIG. 18 .

The pig model is not spontaneous, but the skin architecture most closelyresembles humans. The data suggest that umbilical cord liningmesenchymal stem cell population of the present invention will improvewound healing without the risk of serious adverse side effects. Thesedata thus strongly support the hypothesis that human umbilical cordlining mesenchymal stem cell population as described herein can promotechronic wound healing by suppressing inflammation and promotingangiogenesis. Furthermore, there is clearly no sign of inflammation withthe use of xenogeneic mesenchymal stem cells in either mice or pigs, andtherefore the likelihood that allogeneic mesenchymal stem cells willhave any serious adverse effect in humans is very low.

Experiments Showing that the Mesenchymal Stem Cells as Described Hereinare Effective in Topical Treatments in Humans:

Experiments showing that the mesenchymal stem cells as described hereinare effective in topical treatments in humans have been described in WO2007/046775. In particular, as explained in Examples 23-26 of WO2007/046775 mesenchymal stem cells of the amniotic membrane of theumbilical cord (UCMC) could alleviate full thickness burns (Example 23),partial-thickness wounds (Example 24), non-healing radiation wound(Example 25) as well as non-healing diabetic wound and non-healingdiabetic foot wounds (Example 26). Notably, in accordance with Example 2of WO 2007/046775 mesenchymal stem cells were resuspended in PTT-4medium.

Notably, as depicted in FIGS. 6 b and 6 c the stem cell populationobtained by cultivation when using PTT6 (as used herein) cultivationmedium is significantly more homogenous than the population of cellsobtained by using PTT4 medium (used in WO 2007/046775). Since PTT-4 wasused as medium for mesenchymal stem cells in Examples 23-26 of WO2007/046775 it is clear that the even more homogenous mesenchymal stemcell population isolated after cultivation in PTT-6 (as used herein)will have the same beneficial effects in wound healing applications,such as full thickness burns, partial-thickness wounds, non-healingradiation wound as well as non-healing diabetic wound and non-healingdiabetic foot wounds.

Experiments Showing that the Mesenchymal Stem Cells as Described Hereinare Effective in Topical Treatments in Humans:

This is a planned study of escalating doses of the mesenchymal stem cellpopulation obtained as described herein performed at the University ofColorado Anschutz Medical Campus in Aurora, Colo. The goal of this studyis to determine a safe dose of mesenchymal stem cell population asdescribed herein (human umbilical cord lining mesenchymal stem cells).This is a single-center, dose-escalation study where each of three doselevels will enroll five subjects for a total of fifteen subjects. Thefirst group of 5 patients will receive 100,000 MSC/cm² (skin/wound area)twice per week for 8 weeks. The second group of 5 patients will receive300,000 MSC/cm² twice per week for 8 weeks. The third group of 5patients will receive 500,000 MSC/cm² twice per week for 8 weeks. Thisschedule will continue until either the highest dose is reached, oruntil at least 2 subjects at a dose level have ≥Grade 2 allergicreaction that is suspected to be related to mesenchymal stem cellpopulation as obtained herein or 2 or more subjects at a dose levelexperience an unexpected, treatment-related serious adverse event ordose limiting toxicity within 14 days following the initial dose ofmesenchymal stem cell population as obtained as described herein. All ofthe patients will be evaluated 30 days posttreatment for the productionof anti-HLA antibodies and for wound closure. At the present time, we donot consider production of HLA antibodies to be an absolutecontraindication to a particular dose, but it will factor into ouroverall assessment of safety. This is an open-label study where allsubjects will be taking the study drug and all study personnel will knowthe dose each subject receives. A secondary endpoint of this study willbe significant improvement in the condition of the wound. This endpointwill be based on the rate of wound closure, the percent of wound areasuccessfully closed, and the percent of wounds fully closed, as measuredusing the Silhouette Wound Measurement and Documentation System. Thisdevice is approved by the FDA for this purpose.

Subject Population. Patients with Type I or Type II diabetes withchronic foot ulcers that have not healed after at least 30 days ofconventional therapy and are negative for HLA antibodies to themesenchymal stem cell population as described herein. Patients willcontinue with conventional wound treatment for the first 2 weekscommencing at the time of enrollment, at which time they will havealready been screened for having a diabetic foot ulcer that has nothealed in 30 days. Photodocumentation and measurement of woundparameters will start at this time. Conventional dressing changes willbe performed twice a week for the first 2 weeks, after which mesenchymalstem cell population as described herein will be applied to the wound atthe specified concentrations twice a week. The mesenchymal stem cellpopulation as described herein—treated wounds will also be covered withTegaderm® and a crepe dressing.

Dose Levels. The goal of this study is to determine a safe dose of humanumbilical cord lining mesenchymal stem cells as described herein forfurther study. Patients will be treated with one of three doses: 100,000cells/cm² skin/wound area, 300,000 cells/cm² or 500,000 cells/cm² twicea week for 8 weeks. Each 100,000 cell dose represents 0.1 ml of themesenchymal stem cell population as described herein from a vialcontaining 1 million cells/ml in HypoThermosol.

Dosing Regimen. This is a safety and tolerability study of escalatingdoses of mesenchymal stem cells as described herein. The goal of thisstudy is to determine a safe dose of the human umbilical cord liningmesenchymal stem cells as described herein for further study. Each ofthree dose levels will enroll five subjects. The first group of 5patients will receive 100,000 MSC/cm² skin/wound area twice per week for8 weeks. The second group of 5 patients will receive 300,000 MSC/cm²twice per week for 8 weeks. The third group of 5 patients will receive500,000 MSC/cm² twice per week for 8 weeks. This schedule will continueuntil either the highest dose is reached, or until at least 2 subjectsat a dose level have ≥Grade 2 allergic reaction that is suspected to berelated to mesenchymal stem cells as described herein or 2 or moresubjects at a dose level experience an unexpected, treatment-relatedserious adverse event or dose limiting toxicity within 30 days followingthe initial dose of a mesenchymal stem cell population as describedherein. All of the patients will be evaluated 30 days posttreatment forthe production of anti-HLA antibodies and for degree of wound closure.At the present time, we do not consider production of HLA antibodies tobe an absolute contraindication to a particular dose, but it will factorinto our overall assessment of safety. This is an open-label study whereall subjects will be taking the study drug and all study personnel willknow the dose each subject receives.

Route of Administration. The mesenchymal stem cell population asdescribed herein as described herein are applied topically to debrideddiabetic foot ulcers and held in place by a Tegaderm® bandage.

Dosing Procedure. Following suitable debridement, if needed, the patientis placed in the prone position and the affected leg bent at a 90°angle. This vial of the mesenchymal stem cell population as describedherein is gently swirled to ensure equal distribution of the cells. Theelevated foot is then treated by removing 100,000 (0.1 ml) to 500,000(0.5 ml) cells per cm² from the vial using a sterile syringe and placingit in the center of the wound. The wound is then sealed with a Tegaderm®membrane and gently massaged to distribute the cells evenly. The foot ismaintained elevated for five minutes to allow the cells to settle andattach. The foot is then dressed with a crepe bandage to cover theTegaderm® dressing.

The invention is further characterized by the following items:

1. A method of transporting a stem cell population, the methodcomprising transporting said stem cell population contacted with aliquid carrier, said liquid carrier comprising

i) Trolox;

ii) Na⁺;

iii) K⁺;

iv) Cl⁻;

v) H₂PO₄ ⁻;

vi) HEPES;

vii) Lactobionate;

viii) Sucrose;

ix) Mannitol;

x) Glucose;

xi) Dextran-40;

xii) Adenosine, and

xiii) Glutathione.

2. The method of item 1, wherein the transporting is performed for about7 days or less.3. The method of item 1 or 2, wherein the transporting is performed forabout 6 days, about 5 days, about 4 days, about 3 days, about 2 days,about 1 day or for less than about 1 day.4. The method of any one of the preceding items, wherein thetransporting is performed for about 48 hours or about 24 hours or less.5. The method of any one of the foregoing items, wherein thetransporting is performed at a temperature of about −5° C. to about 15°C.6. The method of any one of the foregoing items, wherein thetransporting is performed at a temperature of about 2° C. to about 8° C.7. The method of any one of the foregoing items, wherein thetransporting is carried out at a temperature of more than about −5° C.,more than about −10° C., more than about −15° C., or more than about−20° C.8. The method of any one of the foregoing items, wherein the stem cellpopulation is transported in a concentration of about 70 million cellsper 1 ml carrier, of about 60 million cells million cells per 1 mlcarrier, of about 50 million cells per 1 ml carrier, of about 40 millioncells per 1 ml carrier, of about 30 million cells per 1 ml carrier, ofabout 20 million cells per 1 ml carrier, of about 10 million cells per 1ml carrier, of about 5 million cells per 1 ml carrier, of about 4million cells per 1 ml carrier, of about 3 million cells per 1 mlcarrier, of about 2 million cells per 1 ml carrier, of about 1 millioncells per 1 ml carrier, of about 0.5 million cells per 1 ml carrier, ofabout 0.1 million cells per 1 ml carrier or of less than 0.1 millioncells per 1 ml carrier.9. The method of item 8, wherein the stem cell population is transportedin a concentration of about 10 million cells per ml carrier to about 1million cells per 1 ml carrier.10. The method of any one of the foregoing items, wherein the stem cellpopulation is an embryonic stem cell population, an adult stem cellpopulation, a mesenchymal stem cell population or an induced pluripotentstem cell population.11. The method of any one of the foregoing items, wherein the stem cellpopulation is a mesenchymal stem cell population.12. The method of any one of the foregoing items, wherein themesenchymal stem cell population is an isolated mesenchymal stempopulation of the amniotic membrane of the umbilical cord.13. The method of item 11 or 12, wherein at least about 90% or morecells of the isolated mesenchymal stem cell population express each ofthe following markers: CD73, CD90 and CD105.14. The method of item 13, wherein at least about 91% or more, about 92%or more, about 93% or more, about 94% or more, about 95% or more, about96% or more, about 97% or more, about 98% or more about 99% or morecells of the isolated mesenchymal stem cell population express each ofCD73, CD90 and CD105.15. The method of any one of items 11-14, wherein at least about 90% ormore, about 91% or more, about 92% or more, about 93% or more, about 94%or more, about 95% or more, about 96% or more, about 97% or more, about98% or more about 99% or more of the isolated mesenchymal stem cellslack expression of the following markers: CD34, CD45 and HLA-DR (HumanLeukocyte Antigen-antigen D Related).16. The method of any one of the foregoing items, wherein the stem cellpopulation is contacted with the carrier before transporting.17. The method of any one of the foregoing items, wherein the stem cellpopulation is contacted with the carrier after its harvest.18. The method of item 17, wherein the stem cell population is contactedwith the carrier about 0 minutes, about 1 minute, about 5 minutes, about10 minutes, about 30 minutes, about 45 minutes, about 60 minutes or alonger time after its harvest.19. The method of item 17 or 18, wherein the harvest comprisesseparating the stem cell population from culture medium.20. The method of item 19, wherein separating is performed bycentrifuging the stem cells within a culture medium and decanting theculture medium.21. The method of any one of the foregoing items, wherein the contactingis performed by suspending the stem cell population in a density ofabout 70 million/ml, of about 60 million/ml, of about 50 million/ml, ofabout 40 million/ml, of about 30 million/ml, of about 20 million/ml, ofabout 10 million/ml, of about 5 million/ml, of about 4 million/ml, ofabout 3 million/ml, of about 2 million/ml, of about 1 million/ml, ofabout 0.5 million/ml, of about 0.1 million/ml or of less than 0.1million cells in the carrier.22. The method of item 21, wherein the contacting is performed bysuspending the stem cell population in a density of about 10 million/mlin the carrier.23. The method of item 21 or 22, wherein the stem cells contacted withthe carrier are aliquoted into vials in a volume of about 50 ml, ofabout 20 ml, of about 10 ml, of about 5 ml, of about 4 ml, of about 3ml, of about 2 ml, of about 1 ml, of about 0.5 ml, of about 0.25 ml orof less than 0.25 ml carrier.24. The method of item 21 or 22, wherein the stem cells contacted withthe carrier are aliquoted into vials in an amount of about 1 ml.25. The method of any one of the foregoing items, wherein the carrier isa transport medium or an excipient.26. The method of any one of the foregoing items, wherein the methoddoes not comprise a thawing or freezing step.27. The method of any one of the foregoing items, wherein the carrierdoes not include a dipolar aprotic solvent, in particular DMSO.28. The method of any one of the foregoing items, wherein at most about50%, about 40%, about 30%, about 20%, about 10% or less than about 10%of the stem cells of the population die during transporting compared tothe amount of viable stem cells before transporting.29. The method of any one of the foregoing items, wherein most of thestem cells in the stem cell population have a cell diameter betweenabout 9 μm and about 20 μm after transporting.30. The method of any one of the foregoing items, wherein most of thestem cells in the stem cell population have a cell diameter betweenabout 12 μm and about 16 μm after transporting.31. The method of any one of the foregoing items, wherein aftertransport the stem cell population secretes about as much TGFbeta-1 asbefore transporting.32. The method of any one of the foregoing items, wherein essentially noPDGF-BB and/or IL-10 is detected before and/or after transporting.33. The method of any one of the foregoing items, wherein aftertransporting the mesenchymal stem cell population secretes about as muchVEGF, PDGF-AA, Ang-1, and/or HGF as before transporting.34. A method of treating a subject having a disease, the methodcomprising topically administering a mesenchymal stem cell population asdefined in any one of items 12-15 to the subject, wherein themesenchymal stem cell population is administered within about 96 hoursfrom the time point the mesenchymal stem cell population has beenharvested.35. The method of item 34, wherein the mesenchymal stem cell populationis applied in a unit dosage of about 20 million cells, of about 15million cells, of about 10 million cells, of about 5 million cells, ofabout 4 million cells, of about 3 million cells, of about 2 millioncells, of about 1 million cells, of about 0.5 million cells, of about0.25 million cells or of less than 0.25 million cells.36. The method of item 34 or 35, wherein the mesenchymal stem cellpopulation is applied in a unit dosage of about 10 million cells.37. The method of any one of items 34-36, wherein the mesenchymal stemcell population is applied within about 72 hours, about 48 hours, about24 hours, about 12 hours, about 6 hours or less from the time point themesenchymal stem cell population has been harvested.38. The method of any one of items 34-37, wherein the disease is a skindisease or a wound.39. The method of item 38, wherein the wound is caused by a burn, abite, a trauma, a surgery, or a disease.40. The method of item 39, wherein the wound is caused by diabeticdisease, wherein the wound is preferably a diabetic wound.41. The method of item 40, wherein the wound is diabetic foot ulcer.42. The method of any of items 34 to 41, wherein the unit dosage ofabout 20 million cells, of about 15 million cells, of about 10 millioncells, of about 5 million cells, of about 4 million cells, of about 3million cells, of about 2 million cells, of about 1 million cells, ofabout 0.5 million cells, of about 0.25 million cells or of less than0.25 million cells is administered once or twice a week.43. The method of item 42, wherein the unit dosage of about 20 millioncells, of about 15 million cells, of about 10 million cells, of about 5million cells, of about 4 million cells, of about 3 million cells, ofabout 2 million cells, of about 1 million cells, of about 0.5 millioncells, of about 0.25 million cells or of less than 0.25 million cells isadministered one of twice a week for a period of time of three weeks, offour weeks, or five weeks or of six weeks, or of seven weeks, or ofeight weeks or of ten weeks or more weeks.44. The method of any one of items 34-43, wherein the mesenchymal stemcell population is applied topically and covered by a film or bandage.45. The method of any one of items 34-44, wherein the mesenchymal stemcell population is applied in a dosage of about 1000 cells/cm² to about5 million cells/cm².46. The method of any one of items 34-45, wherein the mesenchymal stemcell population is applied in a dosage of about 100,000 cells/cm², ofabout 300,000 cells/cm² or of about 500,000 cells/cm².47. The method of any one of items 34-46, wherein the mesenchymal stemcell population is applied once, twice or more times a week.48. The method of any one of items 34-47, wherein the mesenchymal stemcell population is applied for one, two, three, four, five, six, seven,eight, nine, ten, elven weeks or more.49. The method of any one of items 34-48, wherein the mesenchymal stemcell population is applied two times a week for about 8 weeks in adosage of about 100,000 cells/cm², about 300,000 cells/cm² or about500,000 cells/cm².50. The method of any of items 34 to 49, wherein the mesenchymal stemcell population is administered to the subject after separating themesenchymal stem cell population from the carrier.51. The method of item 50, wherein separating the mesenchymal stem cellpopulation from the carrier comprises centrifugation.52. The method of item 50 and 51, separating the mesenchymal stem cellpopulation from the carrier comprises withdrawing the cell populationfrom the vial by means of syringe.53. The method of any of items 34 to 52, comprising administering themesenchymal stem cell population by means of a syringe.54. A unit dosage comprising about 20 million cells, of about 15 millioncells, of about 10 million cells, of about 5 million cells, of about 4million cells, of about 3 million cells, of about 2 million cells, ofabout 1 million cells, of about 0.5 million cells, of about 0.25 millioncells or of less than 0.25 million cells of a mesenchymal stem cellpopulation as defined in any one of items 12-15.55. The unit dosage of item 54, wherein the unit dosage comprises about10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about2, about 1, about 0.5, about 0.25, or about 0.1 million cells.56. The unit dosage of item 54 or 55, wherein the unit dosage comprisesabout 10 million cells.57. The unit dosage of any one of items 54-56, wherein the unit dosagecomprises about 1000 cells to about 5 million cells.58. The unit dosage of any one of items 54-57, wherein the unit dosageis applied in a dosage of about 100,000 cells, 300,000 cells or 500,000cells.59. The unit dosage of any one of items 54-58, wherein the unit dosageis applied topically.60. The unit dosage of any one of items 54-59, wherein the unit dosageis applied topically per cm².61. The unit dosage of any one of items 54-60, wherein the unit dosageis applied once, twice, three times or more times a week.62. The unit dosage of any one of items 54-61, wherein the unit dosageis applied for one, two, three, four, five, six, seven, eight, nine,ten, eleven weeks or more.63. The unit dosage of any one of items 54-62, wherein the unit dosagecomprises of about 100,000 cells, about 300,000 cells or about 500,000cells of any one of items is applied two times a week for 8 weeks.64. The unit dosage of any one of items 54-63, wherein the unit dosageis contained in a 1 ml vial.65. The unit dosage of item 64, wherein 0.1 ml of the vial are applied.66. The unit dosage of any one of items 54 to 65, wherein the cells arein contact with a liquid carrier as defined in item 1.67. The unit dosage of any one of items 54 to 66, wherein the cells arecentrifuged and isolated before administration to a subject.68. The unit dosage of any one of items 54 to 67, wherein the carrier isHypoThermosol™.69. The method of any of items 34 to 53 and the unit dosage of any oneof items 54-68 wherein the cells are viable cells.70. The method of any of items 1 to 33, wherein the carrier isHypoThermosol.71. The use of a liquid carrier for transporting a stem cell population,wherein the liquid carrier comprises

i) Trolox;

ii) Na+;

iii) K+;

iv) Cl−;

v) H2PO4−;

vi) HEPES;

vii) Lactobionate;

viii) Sucrose;

ix) Mannitol;

x) Glucose;

xi) Dextran-40;

xii) Adenosine, and

xiii) Glutathione.

72. The use of item 71, wherein the transporting is performed for about7 days or less.73. The use of item 71 or 72, wherein the transporting is performed forabout 6 days, about 5 days, about 4 days, about 3 days, about 2 days,about 1 day or for less than about 1 day.74. The use of any one of the preceding items 71 to 73, wherein thetransporting is performed for about 48 hours or about 24 hours or less.75. The use of any one of the foregoing items 71 to 74, wherein thetransporting is performed at a temperature of about −5° C. to about 15°C.76. The use of any one of the foregoing items 71 to 75, wherein thetransporting is performed at a temperature of about 2° C. to about 8° C.77. The use of any one of the foregoing items 71 to 76, wherein thetransporting is carried out at a temperature of more than about −5° C.,more than about −10° C., more than about −15° C., or more than about−20° C.78. The use of any one of the foregoing items 71 to 77, wherein the stemcell population is transported in a concentration of about 70 millioncells per 1 ml carrier, of about 60 million cells million cells per 1 mlcarrier, of about 50 million cells per 1 ml carrier, of about 40 millioncells per 1 ml carrier, of about 30 million cells per 1 ml carrier, ofabout 20 million cells per 1 ml carrier, of about 10 million cells per 1ml carrier, of about 5 million cells per 1 ml carrier, of about 4million cells per 1 ml carrier, of about 3 million cells per 1 mlcarrier, of about 2 million cells per 1 ml carrier, of about 1 millioncells per 1 ml carrier, of about 0.5 million cells per 1 ml carrier, ofabout 0.1 million cells per 1 ml carrier or of less than 0.1 millioncells per 1 ml carrier.79. The use of item 78, wherein the stem cell population is transportedin a concentration of about 10 million cells per 1 ml carrier to about 1million cells per 1 ml carrier.80. The use of any one of the foregoing items 71 to 79, wherein the stemcell population is an embryonic stem cell population, an adult stem cellpopulation, a mesenchymal stem cell population or an induced pluripotentstem cell population.81. The use of any one of the foregoing items 71 to 80, wherein the stemcell population is a mesenchymal stem cell population.82. The use of any one of the foregoing items 71 to 81, wherein themesenchymal stem cell population is an isolated mesenchymal stempopulation of the amniotic membrane of the umbilical cord.83. The use of item 81 or 82, wherein at least about 90% or more cellsof the isolated mesenchymal stem cell population express each of thefollowing markers: CD73, CD90 and CD105.84. The use of item 83, wherein at least about 91% or more, about 92% ormore, about 93% or more, about 94% or more, about 95% or more, about 96%or more, about 97% or more, about 98% or more about 99% or more cells ofthe isolated mesenchymal stem cell population express each of CD73, CD90and CD105.85. The use of any one of items 81-84, wherein at least about 90% ormore, about 91% or more, about 92% or more, about 93% or more, about 94%or more, about 95% or more, about 96% or more, about 97% or more, about98% or more about 99% or more of the isolated mesenchymal stem cellslack expression of the following markers: CD34, CD45 and HLA-DR (HumanLeukocyte Antigen-antigen D Related).86. The use of any one of the foregoing items 71 to 85, wherein the stemcell population is contacted with the carrier before transporting.87. The use of any one of the foregoing items 71 to 86, wherein the stemcell population is contacted with the carrier after its harvest.88. The use of item 87, wherein the stem cell population is contactedwith the carrier about 0 minutes, about 1 minute, about 5 minutes, about10 minutes, about 30 minutes, about 45 minutes, about 60 minutes or alonger time after its harvest.89. The use of item 87 or 88, wherein the harvest comprises separatingthe stem cell population from culture medium.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including”, “containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention. Theinvention has been described broadly and generically herein. Each of thenarrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group. Further embodiments of the invention willbecome apparent from the following claims.

When used herein, the term “about” is understood to mean that there canbe variation in the respective value or range (such as pH,concentration, percentage, molarity, number of amino acids, time etc.)that can be up to 5%, up to 10%, up to 15% or up to and including 20% ofthe given value. For example, if a formulation comprises about 5 mg/mlof a compound, this is understood to mean that a formulation can havebetween 4 and 6 mg/ml, preferably between 4.25 and 5.75 mg/ml, morepreferably between 4.5 and 5.5 mg/ml and even more preferably between4.75 and 5.25 mg/ml, with the most preferred being 5 mg/ml. As usedherein, an interval which is defined as “(from) X to Y” equates with aninterval which is defined as “between X and Y”. Both intervalsspecifically include the upper limit and also the lower limit. Thismeans that for example an interval of “5 mg/ml to 10 mg/ml” or “between5 mg/ml and 10 mg/ml” includes a concentration of 5, 6, 7, 8, 9, and 10mg/ml as well as any given intermediate value.

What is claimed is:
 1. A method of transporting a stem cell population,wherein the mesenchymal stem cell population is an isolated humanmesenchymal stem population of the amniotic membrane of the umbilicalcord, the method comprising transporting said stem cell populationcontacted with a liquid carrier, said liquid carrier comprising i)Trolox; ii) Na⁺; iii) K⁺; iv) Cl⁻; v) H₂PO₄ ⁻; vi) HEPES; vii)Lactobionate; viii) Sucrose; ix) Mannitol; x) Glucose; xi) Dextran-40;xii) Adenosine, and xiii) Glutathione.
 2. The method of claim 1, whereinthe transporting is performed for about 6 days, about 5 days, about 4day, about 3 day, about 2 day, about 1 day or for less than about 1 day.3. The method of claim 1, wherein the transporting is performed forabout 48 hours or about 24 hours or less.
 4. The method of claim 1,wherein the transporting is performed at a temperature of about 2° C. toabout 8° C.
 5. The method of claim 1, wherein the stem cell populationis transported in a concentration of about 10 million cells per mlcarrier to about 1 million cells per 1 ml carrier.
 6. The method ofclaim 1, wherein at least about 91% or more, about 92% or more, about93% or more, about 94% or more, about 95% or more, about 96% or more,about 97% or more, about 98% or more about 99% or more cells of theisolated mesenchymal stem cell population express each of CD73, CD90 andCD105.
 7. The method of claim 6, wherein at least about 90% or more,about 91% or more, about 92% or more, about 93% or more, about 94% ormore, about 95% or more, about 96% or more, about 97% or more, about 98%or more about 99% or more of the isolated mesenchymal stem cells lackexpression of the following markers: CD34, CD45 and HLA-DR (HumanLeukocyte Antigen-antigen D Related).
 8. The method of claim 1, whereinthe stem cell population is contacted with the carrier beforetransporting.
 9. The method of claim 8, wherein the stem cell populationis contacted with the carrier after its harvest.
 10. The method of claim9, wherein the contacting is performed by suspending the stem cellpopulation in a density of about 10 million/ml in the carrier.
 11. Themethod of claim 11, wherein the stem cells contacted with the carrierare aliquoted into vials in an amount of about 1 ml.
 12. The method ofclaim 1, wherein the carrier comprises or is HypoThermosol™.
 13. A unitdosage of a mesenchymal stem cell population, wherein the mesenchymalstem cell population is an isolated mesenchymal stem population of theamniotic membrane of the umbilical cord, wherein at least 97% or more ofthe cells of the isolated mesenchymal stem cell population express eachof CD73, CD90 and CD105 and lack expression of each of CD34, CD45 andHLA-DR and wherein the unit dosage comprise about 0.5 million cells toabout 20 million cells.
 14. The unit dosage of claim 12, wherein theunit dose comprises about 15 million cells, about 10 million cells,about 9 million cells, about 8 million cells, about 7 million cells,about 6 million cells, about 5 million cells, about 4 million cells,about 3 million cells, about 2 million cells, or about 1 million cellsof the mesenchymal stem cell population.
 15. The unit dosage of claim13, wherein the unit dosage comprises about 10 million cells.
 16. Theunit dosage of claim 13, wherein the unit dosage is contained in a 1 mlvial.
 17. The unit dosage of claim 13, wherein the unit dose iscontained in a liquid carrier, wherein the liquid carrier comprises i)Trolox; ii) Na⁺; iii) K⁺; iv) Cl⁻; v) H₂PO⁴⁻; vi) HEPES; vii)Lactobionate; viii) Sucrose; ix) Mannitol; x) Glucose; xi) Dextran-40;xii) Adenosine, and xiii) Glutathione.
 18. The unit dosage of claim 17,wherein the carrier comprises HypoThermosol.
 19. The unit dosage ofclaim 13, wherein the cells are viable cells.
 20. The unit dosage ofclaim 13, wherein the unit dose is contained in a volume 1 ml of aliquid carrier.